CN111778316A - Fluorescent probe based on oxidative damage basic group, kit and method for directly detecting DNA methylation - Google Patents
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Abstract
The invention belongs to the field of biological analysis, and relates to a fluorescent probe based on oxidative damage basic groups, a kit and a method for directly detecting DNA methylation. The invention develops a fluorescent probe based on oxidative damage basic groups for the first time, and accurately detects the DNA methylation of a single site through cyclic cutting signal amplification. The hOGG1 enzyme can specifically cleave 8-oxoG base in 8-oxoG/5-mC base pair, but is inactive to 8-oxoG base in 8-oxoG/U base pair. The presence of the target methylated DNA can induce cyclic cleavage of the fluorescent probe with the aid of the hagogg 1 enzyme, resulting in an enhanced fluorescent signal. The method has high sensitivity and good specificity, and can detect single methylationThe position point, the lower detection limit reaches 3.458 × 10‑15And M. Moreover, the method can distinguish the methylation level of 0.01% in a complex DNA mixture system, and can also detect the DNA methylation level in a genome.
Description
Technical Field
The invention belongs to the field of biological analysis, and particularly relates to a fluorescent probe based on oxidative damage basic groups for directly detecting DNA methylation.
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.
Epigenetic modification of DNA can result in heritable changes in gene expression without altering the gene coding sequence. DNA methylation is one of the most important epigenetic events that regulate gene transcription, cell differentiation, and pathogenicity of various diseases including cancer. Mammalian cell DNA methylation occurs almost exclusively at cytosine C-5 of CpG islands. In normal cells, most CpG islands spanning the promoter region are typically unmethylated, and transcription begins because promoter methylation can inhibit promoter activity. Thus, promoter methylation may be another gene inactivation mechanism for chromosomal inactivation and loss-of-function mutations.
The traditional DNA methylation detection method mainly comprises restriction enzyme digestion based on methylation sensitivity, affinity enrichment and sodium bisulfite treatment. However, digestion with restriction enzymes based on methylation sensitivity is prone to false positive results due to incomplete digestion. Affinity enrichment based approaches involve specific antibodies to methylated cytosines or methyl binding domain proteins with affinity to methylated-rich CpG regions, which are very expensive and unstable. Sodium bisulfite treatment deaminates unmethylated cytosine to uracil, while methylcytosine remains unchanged. The sodium bisulfite method distinguishes between methylated cytosines and is currently considered the gold standard for DNA methylation detection. Methylation specific pcr (msp) accurately localizes methylation of CpG islands with extremely high sensitivity, but it presents false positives due to DNA priming errors or incomplete sodium bisulfite conversion. In addition, the heavy methyl and head loop inhibition PCR methods were developed to block amplification of unmethylated DNA. Notably, PCR-based methods rely on stringent primer/template design and precisely optimized thermal cycling. In recent years, new methods have been developed based on sodium bisulfite treatment, such as ligation-dependent assays, Hyperbranched Rolling Circle Amplification (HRCA), secondary isothermal amplification assays, Surface Plasmon Resonance (SPR) -based assays, and Fluorescence Resonance Energy Transfer (FRET) based on Cationic Conjugated Polymers (CCPs) for DNA methylation assays. However, these methods involve complicated ligation processes and DNA polymerase amplification, which limits their widespread use in clinical laboratories. In addition, although some non-PCR methods based on sodium bisulfite treatment, such as electrochemiluminescence, eMethylsorb, have been shown to detect DNA methylation directly, their sensitivity is relatively low.
For example: PCR-based methods typically involve complex procedures (e.g., specific reaction temperatures and cycle numbers), greatly limiting the application of the methods. For example, MS-AP-PCR uses restriction enzymes for methylation analysis, requiring specific restriction sites and radiolabelling. MS-PCR can pinpoint methylation status in CpG islands, but it can only provide qualitative data and not quantitative analysis. MS-qFRET is helpful for directly detecting DNA methylation, but the tail ends of all MSP primers need to be labeled, so the operation is complicated and the price is high. The combination of methylated DNA precipitate with luciferase fusion Zinc fingers (MELZA) requires the construction of MBD clones containing glutathione S-transferase (GST) tags and GST-tagged zif 268-luciferase, a complicated procedure. An electrochemical method based on Graphene Oxide (GO) relates to complex experimental procedures such as synthesis and modification of GO surface, and accumulation of gold nanoparticles (AuNPs) on the surface of a carbon (GC) electrode. Hyperbranched Rolling Circle Amplification (HRCA) involves steps such as ligation, enzymatic digestion, amplification, etc., and is a complex and time-consuming process. The sensitivity of the NESA method is far lower than that of PCR. At present, most methods are suitable for detecting a plurality of methylation sites of the CpG island, but the inventor finds that: few methods are available for direct methylation analysis of a specific single site of genomic DNA.
Disclosure of Invention
In order to overcome the problems, the invention firstly develops a fluorescent probe based on oxidative damage base, and the DNA methylation of a single site is accurately detected through amplifying a cycle cutting signal. The method has high sensitivity and specificityGood in performance, can detect a single methylation site, and the lower detection limit reaches 3.458 × 10-15And M. Moreover, the method can distinguish the methylation level of 0.01% in a complex DNA mixture system, and can also detect the DNA methylation level in a genome.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided an oxidative damage base-based fluorescent probe comprising: 8-oxoguanine 8-oxoG damage base paired with 5-methylcytosine, fluorescent group and quenching group.
The invention designs an 8-oxoguanine (8-oxoG) damaged base modified fluorescent probe, wherein a ROX fluorescent group is modified at the 5 'end, and a BHQ2 quenching group is modified at the 3' end. After bisulfite treatment, the cytosine (C) base in the unmethylated sequence becomes a deoxyribo-diabetic (U) base, while the methylated cytosine C base remains unchanged. The methylated sequences after bisulfite treatment and fluorescent probes are hybridized to form mC/8-oxoG base pairs, hOGG1 glycosylase can recognize and cut 8-oxoG base in the mC/8-oxoG base pairs, so that the signal probes are broken, and fluorescent groups and quenching groups are separated, thereby releasing fluorescent signals to be amplified circularly. While the U base in the bisulfite treated unmethylated sequence pairs with the fluorescent probe to form a U/8-oxoG base pair, hOGG1 glycosylase can hardly cleave the 8-oxoG base in the U/8-oxoG base pair, and thus the signal probe cannot be cleaved and thus a fluorescent molecule cannot be released.
In a second aspect of the present invention, there is provided a kit for directly detecting DNA methylation, comprising: any of the above fluorescent probes.
The kit can directly and accurately perform methylation analysis on a specific single site of the genome DNA. The purpose of signal amplification can be achieved without complex DNA amplification steps, and the sensitivity is high.
In a third aspect of the present invention, there is provided a method for directly detecting DNA methylation, comprising:
bisulfite treating the target DNA to be detected;
hybridizing any one of the fluorescent probes with the bisulfite treated DNA to form double-stranded DNA;
the double-stranded DNA induces the circular cleavage of the fluorescent probe under the assistance of hOGG1 enzyme;
detecting fluorescence signals, and carrying out qualitative/quantitative analysis on DNA methylation.
The invention firstly utilizes the characteristics of DNA damage repair enzyme (human 8-oxoguanine DNA glycosylase, hOGG1) to directly detect DNA methylation. The hOGG1 enzyme can specifically cleave 8-oxoG base in 8-oxoG/5-mC base pair, but is inactive to 8-oxoG base in 8-oxoG/U base pair. The presence of the target methylated DNA can induce cyclic cleavage of the fluorescent probe with the aid of the hagogg 1 enzyme, resulting in an enhanced fluorescent signal.
The invention has the beneficial effects that:
(1) the invention skillfully designs a fluorescent probe based on damaged bases, and directly detects DNA methylation by utilizing the characteristics of DNA damage repair enzyme, which is known for the first time. The hOGG1 enzyme can specifically cleave 8-oxoG base in 8-oxoG/5-mC base pair, but is inactive to 8-oxoG base in 8-oxoG/U base pair. The presence of the target methylated DNA can induce cyclic cleavage of the fluorescent probe with the aid of the hagogg 1 enzyme, resulting in an enhanced fluorescent signal.
(2) The invention directly and accurately carries out methylation analysis on a specific single site of the genome DNA.
(3) The invention can achieve the purpose of signal amplification without complex DNA amplification steps and has high sensitivity.
(4) Binding of the fluorescent probe of the present invention to hOGG1 can distinguish methylated cytosine from unmethylated cytosine;
(5) the fluorescent probe can directly detect DNA methylation under isothermal conditions without strict primer/template design, any thermal cycle and connection procedures, thereby greatly simplifying experimental procedures;
(6) the hOGG1 can induce the cyclic cleavage of the fluorescent probe, thereby effectively amplifying the signal and greatly improving the detection sensitivity.
(7) The method is simple, strong in practicability and easy to popularize.
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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 direct detection of DNA methylation by a fluorescent probe modified based on 8-oxoguanine (8-oxoG) damaged base in example 1 of the present invention. The method comprises three steps: (1) bisulfite treatment of target DNA to be detected (2) hybridization of signaling probes to bisulfite treated DNA, and (3) hOGG 1-induced cyclic cleavage of signaling probes.
FIG. 2 is an analytical map of example 1 of the present invention, (A) native polyacrylamide gel electrophoresis (PAGE) analysis. M is Marker; lane 1:1.6U hOGG1+500nM methylated MLH1 DNA/signal probe hybrid; lane 2:1.6UhOGG1+500nM unmethylated DNA/signal probe hybrid; lane 3:500nM methylated MLH1 DNA/signalprobe hybrid. (B) Fluorescence emission spectra in the presence of 100nM unmethylated DNA (black line) and in the presence of 100nM methylated DNA (red line).
FIG. 3 is a graph showing the relationship between the fluorescence intensity and the concentration of methylated DNA in example 1 of the present invention. (A) Fluorescence spectra of methylated DNA at different concentrations. (B) Fluorescence intensity at 605nm was related to different concentrations of methylated DNA. (C) Fluorescence intensity at 605nm and methylated DNA concentration of 10-13M to 10-9Log linear relationship in the M range (D) fluorescence intensity at 605nm with methylated DNA concentration at 2 × 10-9M to 10-7Log linear relationship in the M range. Error bars show the standard deviation of three independent experiments.
FIG. 4 shows the detection of the level of methylated DNA in the mixed system of example 1 of the present invention. (A) Fluorescence spectra corresponding to different ratios of DNA methylation. (B) The methylation level detected by the method in the mixed system is linearly related to the methylation level actually added. Error bars represent standard deviations of three independent experiments.
FIG. 5 shows fluorescence response signals of methylated MLH1 sequence (MLH1-mC, red), unmethylated MLH1 sequence (MLH1-C, green), methylated arbitrary sequence 1(random1, lake blue), and unmethylated arbitrary sequence 2(random2, yellow) in example 1 of the present invention. All the above sequences were at a concentration of 100 nM. Error bars represent standard deviations of three independent experiments.
FIG. 6 is the detection of methylation in the genome of the cell of example 1 of the present invention. The genomic DNA mass was 2ng and the error bars represent the standard deviation of three independent experiments.
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.
Interpretation of terms
In this application, the fluorescent probe is also called a signaling probe;
short for hOGG1 glycosylase: hOGG 1;
the english name for the ROX fluorophore is: Carboxy-X-Rhodamine;
the english name for the BHQ2 quenching group is: black Hole quench.
The invention develops a fluorescent probe based on oxidative damage basic groups for the first time, and accurately detects the DNA methylation of a single site through cyclic cutting signal amplification. An 8-oxoguanine (8-oxoG) damaged base modified fluorescent probe is designed, a ROX fluorescent group is modified at the 5 'end, and a BHQ2 quenching group is modified at the 3' end.
The choice of the fluorophore and the quencher is not particularly limited, and in some embodiments, the 5' end of the fluorescent probe is modified with the ROX fluorophore. The 3' end of the fluorescent probe is modified with a BHQ2 quenching group. The signal probe has good quenching effect and low background signal.
In some embodiments, the sequence of the fluorescent probe is: ROX-ATC CGC TCT TCC T-BHQ2, where the underlined bold G base indicates an 8-oxoG modification. The fluorescent probe can be hybridized with unmethylated DNA treated by bisulfite to form double-stranded DNA containing U/8-oxoG, and then is recognized and cut by hOGG1 glycosylase to release a fluorescent signal.
The present invention also provides a kit for direct detection of DNA methylation comprising any of the above-described fluorescent probes, wherein after bisulfite treatment, cytosine (C) bases in the unmethylated sequence become deoxyribo-diabetic (U) bases while methylated cytosine C bases remain unchanged, the bisulfite treated methylated sequence hybridizes with the above-described fluorescent probes to form mC/8-oxoG base pairs, hOGG1 glycosylase recognizes and cleaves 8-oxoG bases in the mC/8-oxoG base pairs, cleaving the signal probe, separating the fluorophore and the quencher, thereby releasing fluorescent signal for cyclic amplification, and the U bases in the bisulfite treated unmethylated sequence pair with the fluorescent probes to form U/8-oxoG base pairs, hOGG1 glycosylase hardly cleaves 8-oxoG bases in the U/8-oxoG base pairs, thus the signal probe cannot be cleaved, thereby not releasing fluorescence, the method has high sensitivity, good specificity, can detect a single methylation lower limit, 3.458 × 10.18.18.- 15And M. Moreover, the method can distinguish the methylation level of 0.01% in a complex DNA mixture system, and can also detect the DNA methylation level in a genome.
In some embodiments, the kit further comprises: bisulfite, hOGG1 glycosylase. The DNA is methylated using bisulfite, which in some embodiments is sodium bisulfite. The use of hOGG1 to specifically recognize and cleave the 8-oxoG base in the mC/8-oxoG base pair in the methylated sequence and signal probe hybrid, hardly cleaves the 8-oxoG base in the U/8-oxoG base pair in the unmethylated sequence.
The invention also provides a method for directly detecting DNA methylation, which comprises the following steps:
bisulfite treating the target DNA to be detected;
hybridizing any one of the fluorescent probes with the bisulfite treated DNA to form double-stranded DNA;
the double-stranded DNA induces the circular cleavage of the fluorescent probe under the assistance of hOGG1 enzyme;
detecting fluorescence signals, and carrying out qualitative/quantitative analysis on DNA methylation.
The principle of the detection is that after bisulfite treatment, cytosine (C) bases in the unmethylated sequences become deoxyribo-diabetic (U) bases, while methylated cytosine C bases remain unchanged, the bisulfite treated methylated sequences hybridize with the fluorescent probes to form mC/8-oxoG base pairs, hOGG1 glycosylase can recognize and cleave 8-oxoG bases in mC/8-oxoG base pairs, cleaving the signaling probes, separating the fluorescent groups from the quenching groups, thereby releasing fluorescent signals to be amplified cyclically, while the U bases in the bisulfite treated unmethylated sequences pair with the fluorescent probes to form U/8-oxoG base pairs, hOGG1 glycosylase can hardly cleave 8-oxoG bases in U/8-oxoG base pairs, thus the signaling probes can not be cleaved, thereby not releasing fluorescence-15And M. Moreover, the method can distinguish the methylation level of 0.01% in a complex DNA mixture system, and can also detect the DNA methylation level in a genome.
The research of the application finds that: as the concentration of methylated DNA increases, the fluorescence signal increases. In some embodiments, the concentration of methylated DNA is 10-13M to 10-9M in range and 2 × 10-9M to 10-7In the M range, the fluorescent intensity and the concentration logarithm of the methylated DNA are in a linear relationship, so that the quantitative detection of the methylated DNA is realized.
In some embodiments, the concentration of methylated DNA is at 10-13M to 10-9In the range of M, the regression equation is F1466 +101.7log10C, F is the fluorescence intensity at 605nm, C is the concentration of methylated DNA, the detection limit is 3.458 × 10-15M (3.458 fM). Compared with the gold nanoparticle-based colorimetric analysis (0.1 mu M), the sensitivity is improved by 8 orders of magnitude, compared with the electrochemical luminescence analysis (1.8nM), the sensitivity is improved by 6 orders of magnitude, and compared with the fluorescence method of double isothermal amplification (0.78pM), the sensitivity is improved by 2 orders of magnitude.
In some embodiments, the concentration of methylated DNA is 2 × 10-9M to 10-7In the range of M, the regression equation is F12506 +1367log10C, F is the fluorescence intensity at 605nm, C is the methylated DNA concentration. The fluorescent signal amplification effect is good, and the sensitivity is high.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
Example 1:
cell culture and preparation of genomic DNA samples: human colon cancer cells (SW480 cells) were cultured in Dulbecco's modified eagle's medium DMEM containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin at 37 ℃ with 5% CO2Is cultured in a humid environment. Genomic DNA was extracted according to the QIAamp DNA mini kit (QIAGEN) instructions. Cells were first lysed extensively with lysis buffer containing proteinase K, incubated at 56 ℃ for 10min, and then an equal volume of 100% ethanol was added. The genomic DNA was bound to a QIAamp column, washed twice and then eluted to collect pure genomic DNA. Genomic DNA concentration was determined using a NanoDrop 2000 spectrophotometer. 500 pg-2. mu.g of genomic DNA was treated with bisulfite according to the EZ-DNA methylation-gold kit instructions.
Bisulfite treatment of DNA: bisulfite treatment of DNA was performed according to the instructions of the EZ-DNA methylation-gold kit.
1. mu.L of CT (conversion reagent) conversion reagent was added to 20. mu.L of the DNA sample in the PCR tube.
2. The sample tube was placed in a PCR instrument and the following steps were performed (1) at 98 ℃ for 10 minutes (2) at 64 ℃ for 2.5 hours.
3. 600 μ L M-Binding Buffer was added to the separation column and the separation column was placed in the collection tube provided.
4. The sample from step 2 was loaded into a column containing M-Binding Buffer. The lid was closed and the column was inverted several times to mix the solution.
5. Centrifuge at full speed (>10,000g) for 30 seconds and discard the effluent.
6. 100 μ L M-Wash Buffer was added to the column and centrifuged at full speed for 30 seconds.
7. 200 μ L M-Desulphosphorylation Buffer was added to the column and allowed to stand at room temperature for 20 minutes. After incubation, the cells were centrifuged at full speed for 30 seconds.
8. 200 μ L M-Wash Buffer was added to the column. Centrifuge at full speed for 30 seconds. An additional 200. mu. L M-WashBuffer was added and centrifuged again for 30 seconds.
9. The column was placed into a 1.5mL microcentrifuge tube. 10 μ L M-Elution Buffer was added directly to the column matrix. The DNA was eluted by centrifugation at full speed for 30 seconds.
And (3) DNA methylation detection: different concentrations of bisulfite treated synthetic target sequence methylated DNA were added to 20. mu.L of a reaction containing 0.5. mu.M signal probe, 1.6U hOGG1, 1 XNEB buffer 2, 1. mu.g BSA, followed by incubation at 40 ℃ for 60 min in the absence of light.
Gel electrophoresis analysis: 12% Polyacrylamide gel electrophoresis (PAGE) was performed in 1% TBE buffer (9mM Tris-HCl, 9mM boric acid, 0.2mM EDTA, pH 7.9) at 110V constant voltage for 50min at room temperature. The gel electrophoresis images were imaged by a Bio-Rad ChemiDoc MP imaging system.
Fluorescence spectrum analysis: mu.L of the reaction product was diluted with ultrapure water to a final volume of 60. mu.L, and the fluorescence spectrum was measured with Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan). ROX fluorescence was measured at an excitation wavelength of 580nm, and quantitative analysis was performed using the fluorescence intensity at 605 nm.
Detection of methylation levels in the genome: to increase the sensitivity of the detection, the bisulfite treated genome is first PCR amplified and then detected. PCR reaction 25. mu.L containing genomic DNA, 0.4. mu.M forward primer, 0.4. mu.M reverse primer, 400. mu.M dNTPs (dATP, dCTP, dGTP and dUTP in a ratio of 1:1:1:1), 1 × PCR buffer (10mM Tris-HCl,50mM KCl,1mM MgCl2pH 8.3) and 2.5U Taq DNA polymerase PCR reaction conditions 95 ℃ 5 min, 94 ℃ 30 sec, 56 ℃ 30 sec, 72 ℃ 30 sec, 40 cycles, 72 ℃ 5 min, then 8U Lambda exonuclease, 3. mu.L 10 × Lambda buffer was added to the above reaction to a total volume of 30. mu.L, incubated at 37 ℃ for 60 min followed by incubation at 85 ℃ for 10min, finally 10. mu.L of the above cleavage product was added to 20. mu.L reaction containing 0.5. mu.M signal probe, 1.6U hOGG1, 1 × NEB buffer 2, 2. mu.g BSA and incubated at 40 ℃ protected from light for 60 min.
Detection principle of DNA methylation: FIG. 1 shows the principle of direct detection of DNA methylation using a fluorescent probe modified with 8-oxoguanine (8-oxoG) damaged bases. The method comprises three steps: (1) bisulfite treatment of methylated and unmethylated DNA to be detected, (2) hybridization of the signaling probe to bisulfite treated methylated and unmethylated DNA, respectively, to form a duplex, (3) cyclic cleavage of the signaling probe by hOGG1 glycosylase to produce an enhanced fluorescent signal. Normal unmethylated cytosine C after bisulfite treatment will become uracil U, while methylated 5-methylcytosine (5-mC) will remain the C base. We designed a signaling probe that pairs perfectly complementary to the methylated DNA sequence to be detected, where the G base pairing with 5-mC was designed as an 8-oxoguanine (8-oxoG) damaged base, the 5 'end of the signaling probe was modified with a ROX fluorophore, and the 3' end was modified with a BHQ2 quencher. The bisulfite treated methylated DNA hybridizes with the added signal probe to form double-stranded DNA containing mC/8-oxoG, while the bisulfite treated unmethylated DNA may hybridize with the signal probe to form double-stranded DNA containing U/8-oxoG. The hOGG1 glycosylase can recognize and cleave the 8-oxoG base in the mC/8-oxoG base pair, generate a gap, cause the signal probe to break and dissociate from the double strand, release the fluorescent molecule ROX, generate a fluorescent signal, and release the methylated sequence matched with the signal probe. The released methylated DNA can then hybridize with a new signaling probe to initiate the hybridization-cleavage-hybridization cycle, generating a large amount of ROX fluorescent signal. The hOGG1 glycosylase has extremely low recognition capability and cutting capability on U/8-oxoG base pairing formed by hybridization of unmethylated DNA and a signal probe, so that the signal probe is hardly cut by the hOGG1 glycosylase, and ROX fluorescent molecules are not released, and a remarkable fluorescent signal is not generated.
1. And (5) verifying the feasibility of the method.
To verify the feasibility of the method, a sequence in the promoter sequence of the tumor suppressor MLH1 was selected as a model. After bisulfite treatment, gel electrophoresis experiments were first used to verify whether hOGG1 glycosylase could recognize and cleave the 8-oxoG base in the mC/8-oxoG base pair, but not the 8-oxoG base in the U/8-oxoG base pair. When methylated MLH1 DNA was present with the signaling probe hybrid and hOGG1, it was seen that the signaling probe was cleaved, releasing the ROX fluorescent molecule (4nt) (FIG. 2A, lane 1). When a hybrid of unmethylated DNA with signaling probe and hOGG1 were present, it was seen that the signaling probe was barely cleaved (FIG. 2A, lane 2), and its band was identical in size to that in the absence of hOGG1 (FIG. 2A, lane 3). This result indicates that hOGG1 can cleave the 8-oxoG base of the mC/8-oxoG base pair in the methylated DNA and signaling probe hybrid, thereby causing cleavage of the signaling probe; but not the 8-oxoG base of the U/8-oxoG base pair. In addition, the fluorescence spectrum experiment is used for verifying whether the methylated DNA sequence treated by bisulfite can be detected by the method. A distinct emission peak at wavelength 605nm can be seen when methylated DNA sequences are present (FIG. 2B, red line); when unmethylated DNA sequences were present, there was no significant emission peak at wavelength 605nm (FIG. 2B, black line). This result is sufficient to demonstrate that the method can clearly distinguish between methylated and unmethylated DNA.
2. And (4) analyzing the sensitivity of the method.
To investigate the methylation assay performance of this method, we tested methylated DNA at different concentrations under optimal conditions. FIG. 3A shows the fluorescence spectrum as a function of methylated DNA concentration. The fluorescence signal increases with increasing methylated DNA concentration from 0 to 100 nM. To perform quantitative determinationFor analysis, we read the fluorescence intensity at 605 nm. FIG. 3B shows the fluorescence intensity at 605nm as a function of different concentrations of methylated DNA. It can be seen that as the concentration of methylated DNA increases, the fluorescence signal increases. Furthermore, the concentration of methylated DNA was 10-13M to 10-9M range (FIG. 3C) and 2 × 10-9M to 10-7Fluorescence intensity in the M range (FIG. 3D) is logarithmically linear with concentration. When the concentration of methylated DNA is 10-13M to 10-9In the range of M, the regression equation is F1466 +101.7log10C(R20.9929) when the concentration of methylated DNA was 2 × 10-9M to 10-7In the range of M, the regression equation is F12506 +1367log10C(R20.9930), (F is the fluorescence intensity at 605nm, C is the concentration of methylated DNA) the limit of detection was calculated to be 3.458 × 10 based on the mean signal plus 3 standard deviations of the control group-15M (3.458 fM). Compared with the gold nanoparticle-based colorimetric analysis (0.1 mu M), the sensitivity is improved by 8 orders of magnitude, compared with the electrochemical luminescence analysis (1.8nM), the sensitivity is improved by 6 orders of magnitude, and compared with the fluorescence method of double isothermal amplification (0.78pM), the sensitivity is improved by 2 orders of magnitude. The high sensitivity of this method is mainly due to the following aspects: (1) the signal probe has good quenching effect and low background signal; (2) hOGG1 specifically recognizes and cleaves 8-oxoG base in mC/8-oxoG base pair in the methylated sequence and signal probe hybrid, and hardly cleaves 8-oxoG base in U/8-oxoG base pair in the unmethylated sequence; (3) the hOGG1 can cyclically cleave the signaling probe, allowing amplification of the fluorescent signal.
3. Detection of DNA methylation levels in the mixed system.
To evaluate whether this method can accurately detect methylated DNA in a mixed system of methylated and unmethylated DNA, we prepared mixtures of methylated and unmethylated DNA in different ratios. The total concentration of methylated and unmethylated DNA was 100nM, and the mixtures contained 0.01%, 0.1%, 1%, 5%, 10%, 20%, 50%, and 100% methylated DNA, respectively. As shown in FIG. 4A, as the proportion of methylated DNA in the mixture increases, the fluorescence intensity also increases. This method can even distinguish methylation levels as low as 0.01% in mixed systems, which is superior to most methods currently used for DNA methylation analysis, such as MALDI-Mass Spectrometry (5%), FRET method based on Quantum dots (1%), cationic conjugated polyelectrolyte method (1%). The level of methylated DNA actually detected by the method is the value obtained by dividing the concentration of methylated DNA measured by the method by the total concentration of methylated DNA and unmethylated DNA. The measured DNA methylation level was linear with the actual DNA methylation level added (FIG. 4B). The regression equation was 0.9968X +0.011 with a correlation coefficient of 0.9995, where Y is the detected DNA methylation level (%) and X is the added DNA methylation level (%). These results indicate that the proposed method can specifically and sensitively detect methylated DNA without interference from large amounts of unmethylated DNA sequences.
4. And (5) verifying the specificity of the method.
To verify the specificity of the proposed method, we detected the methylated MLH1 sequence (FIG. 5, red), the unmethylated MLH1 sequence (MLH1-C, FIG. 5, green), the methylated arbitrary sequence 1(random1, FIG. 5, lake blue), the unmethylated arbitrary sequence 2(random2, FIG. 5, yellow) respectively with signaling probes that specifically detected the methylated MLH1 sequence (MLH 1-mC). As can be seen from FIG. 5, a strong fluorescence signal is generated when the methylated MLH1 sequence (MLH1-mC) is present, while almost no fluorescence signal is generated when the unmethylated MLH1-C sequence, the methylated arbitrary sequence 1(random 1), and the unmethylated arbitrary sequence 2(random 2) are present. The method has good specificity, can distinguish methylated DNA from non-methylated DNA, and can accurately detect the methylation of a specific site by designing a specific signal probe.
5. Detection of methylation in the genome of the cell.
Cytosine methylation of the hMLH1 promoter region is a common phenomenon in colorectal cancer. We further detected methylation of CpG sites in the MLH1 promoter region (cancer suppressor gene) of human colon cancer cells (SW480 cells) using this fluorescent probe. Genomic DNA extracted from SW480 cells was treated with sodium bisulfite and detected with a fluorescent probe. As shown in FIG. 6, the fluorescence intensity increased in a concentration-dependent manner, and the fluorescence intensityHas a good linear relationship with the amount of genomic DNA. The regression equation is that F is 123.3+2.87A (R)20.995), where F is the fluorescence intensity at 605nm and a is the amount of genomic DNA. The detection limit is 26.72ng calculated by adding 3 times of standard deviation to the average blank signal, which is superior to SPR method based on PCR amplification (50ng), and the fluorescent probe can sensitively detect the methylation state of the cancer cells.
TABLE 1 oligonucleotide sequences
The underlined C base indicates a 5-methylcytosine (5-mC) modification. In the fluorescent probe, the underlined bold G bases indicate 8-oxoG modification. Asterisks indicate thio modifications and P indicates phosphate modifications.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. 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. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. A fluorescent probe based on oxidatively damaged bases, comprising: 8-oxoguanine 8-oxoG damage base paired with 5-methylcytosine, fluorescent group and quenching group.
2. The oxidative damage base-based fluorescent probe of claim 1, wherein the 5' end of the fluorescent probe is modified with a ROX fluorophore.
3. The oxidative damage base-based fluorescent probe of claim 1, wherein the 3' end of the fluorescent probe is modified with a BHQ2 quenching group.
4. The oxidative damage base-based fluorescent probe of claim 1, wherein the sequence of the fluorescent probe is: ROX-ATC CGC TCT TCC T-BHQ2, where the underlined bold G base indicates an 8-oxoG modification.
5. A kit for directly detecting DNA methylation, comprising: the fluorescent probe of any one of claims 1-4.
6. The kit for directly detecting DNA methylation according to claim 5, wherein the kit further comprises: bisulfite, hOGG1 glycosylase.
7. A method for directly detecting DNA methylation, comprising:
bisulfite treating the target DNA to be detected;
hybridizing the fluorescent probe of any one of claims 1-4 to bisulfite treated DNA to form double stranded DNA;
the double-stranded DNA induces the circular cleavage of the fluorescent probe under the assistance of hOGG1 enzyme;
detecting fluorescence signals, and carrying out qualitative/quantitative analysis on DNA methylation.
8. The method for directly detecting DNA methylation according to claim 7, wherein the concentration of methylated DNA is 10-13M to 10-9M in range and 2 × 10-9M to 10-7In the M range, the fluorescence intensity is logarithmically linear with the concentration of methylated DNA.
9.The method for directly detecting DNA methylation according to claim 7, wherein the concentration of methylated DNA is 10-13M to 10-9In the range of M, the regression equation is F1466 +101.7log10C, F is the fluorescence intensity at 605nm, C is the methylated DNA concentration.
10. The method for directly detecting DNA methylation according to claim 7, wherein the concentration of methylated DNA is 2 × 10-9M to 10-7In the range of M, the regression equation is F12506 +1367log10C, F is the fluorescence intensity at 605nm, C is the methylated DNA concentration.
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