CN114507713A - Bsu polymerase mediated fluorescence coding sensor and application thereof in 8-oxo-7, 8-dihydroguanine detection - Google Patents
Bsu polymerase mediated fluorescence coding sensor and application thereof in 8-oxo-7, 8-dihydroguanine detection Download PDFInfo
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Abstract
The invention belongs to the technical field of fluorescence detection, and provides a Bsu polymerase mediated fluorescence coding sensor and application thereof in 8-oxo-7, 8-dihydroguanine detection. The fluorescence coding sensor at least comprises a DNA capture probe, Bsu polymerase and dATP modified by a fluorescent group. The Bsu polymerase-mediated fluorescence coding sensor designed by the invention can accurately determine the oxidative damage on telomere specific sites by utilizing the high extension capability and high differentiation capability of Bsu polymerase; background signals are effectively reduced by utilizing the high separation efficiency of the magnetic beads; meanwhile, the single molecule detection sensitivity is extremely high, the sample consumption is low, and in addition, OG in genomic DNA extracted from the HeLa cells treated by the hydrogen peroxide can be accurately quantified, so that the method has great potential in the aspects of disease specific gene damage research and early clinical diagnosis.
Description
Technical Field
The invention belongs to the technical field of fluorescence detection, and particularly relates to a Bsu polymerase mediated fluorescence coding sensor and application thereof in 8-oxo-7, 8-dihydroguanine detection.
Background
The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Reactive Oxygen Species (ROS) are produced by endogenous factors (e.g., cellular metabolism), exogenous stimuli (e.g., ultraviolet radiation and ionizing radiation), and environmental toxins. ROS can oxidize cellular macromolecules such as nucleic acids to produce single base damage and DNA strand breaks that can alter gene expression and regulation, impair genomic stability, promote aging and carcinogenesis. Guanine (G) is most easily oxidized in 4 DNA bases because of its lowest redox potential. One of the major oxidation products of G is 8-oxo-7, 8-dihydroguanine (OG), which can occur in any part of chromosomal DNA, including telomeres that protect the ends of chromosomes. Furthermore, the oxidation potential of G decreases upon stacking of single chains, promoting hole transfer by GGG triplet traps, and acting as preferential sites for redox reactions. OG is associated with changes in telomere length and integrity and can lead to telomere shortening, dysfunction, cellular senescence, and even cancer. Therefore, identification of OG in telomeres is crucial to studying genotoxicity.
Conventional OG detection methods include High Performance Liquid Chromatography (HPLC) and HPLC combined with gas chromatography-mass spectrometry (GC-MS), but they all require complicated instruments and cumbersome procedures, or incubation at high temperatures for long periods of time for gas chromatography-mass spectrometry analysis. Single cell gel electrophoresis is cost effective for the detection of cellular OG levels, but does not provide quantitative information. In recent years, enzymes with good specificity for OG have been used to initiate the DNA base excision repair pathway and remove OG, leaving an apurinic site that can be converted to DNA fragmentation by a cleaving enzyme. Synthetic DNA fragmentation can be measured by PCR and next generation sequencing, but the inventors found that these methods involve multiple enzymatic reactions and cannot quantify OG levels in a particular sequence. Therefore, sensitive detection of low abundance OG levels in a particular sequence remains a significant challenge.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a Bsu polymerase mediated fluorescence coding sensor and application thereof in 8-oxo-7, 8-dihydroguanine detection. The Bsu polymerase-mediated fluorescence coding sensor designed by the invention can accurately determine the oxidative damage on telomere specific sites by utilizing the high extension capability and high differentiation capability of Bsu polymerase; background signals are effectively reduced by utilizing the high separation efficiency of the magnetic beads; meanwhile, the single molecule detection sensitivity is extremely high, the sample consumption is low, and in addition, OG in genomic DNA extracted from the HeLa cells treated by the hydrogen peroxide can be accurately quantified, so that the method has great potential in the aspects of disease specific gene damage research and early clinical diagnosis.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
in a first aspect of the invention, a Bsu polymerase-mediated fluorescence-encoded sensor is provided, the fluorescence-encoded sensor comprising at least a DNA capture probe, a Bsu polymerase and a fluorophore-modified dATP;
wherein the DNA capture probe has a complementary region hybridized with a sequence to be detected, so that the DNA capture probe can be hybridized with the sequence to be detected to form a partial double-stranded structure; but it does not contain a base sequence that is complementary paired to the OG site.
The 5' end of the DNA capture probe is modified with biotin so as to be combined with Magnetic Beads (MB) coated with streptavidin;
since Bsu polymerase has the greatest (34.1-fold) ability to discriminate OG from G in single nucleotide primer extension reactions among all polymerases, Bsu polymerase was selected for telomere-OG discrimination and encoding.
In the fluorophore modified dATP, the fluorophore is not particularly limited, and in one embodiment of the present invention, the fluorophore may be Cy5, and Cy5-dATP is embedded into the capture probe at the OG complementary position by using Bsu polymerase to generate Cy 5-labeled double-stranded DNA, which can be immobilized on the MB surface by the specific action of streptavidin and biotin to form an MB-capture probe-Cy 5 complex. Thus, the fluorescence-encoded sensor further comprises streptavidin-coated magnetic beads.
Further, after magnetic separation, exonuclease iii (exoiii) was added to digest the Cy 5-labeled capture probe into mononucleotides, releasing Cy5 fluorescent molecules from the MB surface. The released Cy5 fluorescent molecules can be simply counted by single molecule detection. Thus, the fluorescence-encoded sensor further comprises exonuclease III.
In a second aspect of the invention, there is provided the use of a fluorescence-encoded sensor as described above for the detection of 8-oxo-7, 8-dihydroguanine.
In a third aspect of the invention, there is provided a method for detecting 8-oxo-7, 8-dihydroguanine, said method comprising detecting with a fluorescence-encoded sensor as described above.
In a fourth aspect of the present invention, there is provided the use of the fluorescence-encoded sensor and/or the detection method described above in 8-oxo-7, 8-dihydroguanine-related drug screening and/or 8-oxo-7, 8-dihydroguanine analysis of a biological sample.
Although the present invention provides a fluorescence-encoded sensor and a detection method for detecting 8-oxo-7, 8-dihydroguanine as an example, it is also conceivable to use the fluorescence-encoded sensor and the detection method for detecting other oxidized products of nucleic acid based on the concept of the present invention, and therefore, the present invention is considered to fall within the scope of the present invention.
The beneficial technical effects of one or more technical schemes are as follows:
1. the detection time of the technical scheme is very short (about 1h), the method is very simple, and no nucleic acid amplification or specific restriction enzyme recognition reaction is needed;
bsu polymerase can selectively incorporate Cy5-dATP into the relative site of OG, and has good specificity;
MBs can effectively capture, separate and concentrate telomeres from a total genome, so that the analysis speed is remarkably improved, and the detection sensitivity is improved;
4. the introduction of the single molecule detection greatly reduces the sample consumption and improves the detection sensitivity, thereby having good value of practical application.
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 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 the principle of Bsu polymerase mediated fluorescence encoding for detecting OG in the present invention.
FIG. 2 is a chart relating to feasibility analysis in an embodiment of the present invention. Wherein (A) base pairs via hydrogen bonds between OG and adenine. The dotted line represents a hydrogen bond. (B) There is no base pairing between OG and adenine. The dotted line represents a hydrogen bond. (C) Bsu polymerase mediated fluorescence encoded product was electrophoresed using a 20% non-denaturing polyacrylamide gel. Lane 1, 250nM telomere-OG; lane 2, 250nM capture probe; lane 3, 250nM normal telomeres +250nM capture probe + 2.5. mu.M Cy5-dATP +0.0625U Bsu polymerase; lane 4, 250nM telomere-OG +250nM + capture probe + 2.5. mu.M Cy5-dATP +0.0625U Bsu polymerase. (D) Cy5 fluorescence signals were detected from 250nM telomere-OG and 250nM normal telomeres.
FIG. 3 is a correlation diagram of single molecule imaging in an embodiment of the present invention. Wherein (A) single molecule fluorescence imaging of Cy5 in the presence of 250nM normal telomeres. The scale bar is 5 μm. (B) Single molecule fluorescence imaging of Cy5 in the presence of 250nm telomeric g. The scale bar is 5 μm. (C) Change in oxidative damage counts versus Cy5 at different concentrations. (D) The linear relationship between Cy5 counts and the logarithm of the oxidative damage concentration ranged from 0.5aM to 5 pM.
FIG. 4 is a graph of sensitivity detection correlation in an embodiment of the present invention. Wherein (A) is a fluorescence image before a photobleaching process. The scale bar is 1 μm. (B) The fluorescence intensity over time showed a photobleaching step for a single Cy5 fluorescent spot.
FIG. 5 is a diagram showing the correlation between specificity analysis in the examples of the present invention. Wherein (A) Cy5 counts were determined at 250nM normal telomeres, 250nM KARS, 250nM random DNA and 250nM telomere-OG, respectively. (B) Cy5 counts were measured at 250nM normal telomeres, 250nM DNA-1, 250nM DNA-2 and 250nM telomere-OG, respectively.
FIG. 6 is a diagram of OG detection in a complex system according to an embodiment of the present invention. Wherein (A) the linear relationship between the added OG and the measured OG level. The total concentration of normal telomeres and telomere-OG was 250nM and the mixtures contained 0.01%, 0.1%, 1%, 5%, 10% and 100% telomere-OG, respectively. (B) After treatment with hydrogen peroxide of different concentrations, the telomeres of HeLa cells are changed by oxidative damage. The amount of genomic DNA derived from HeLa cells was 10 ng. (C) Cy5 counts produced by lysis buffer, HL-7702 cells, A549 cells and HeLa cells were determined. Human cell lines were treated with 1000. mu.M hydrogen peroxide. The amount of genomic DNA derived from each cell was 10 ng. (D) Cy5 counts were linear with the log of genomic DNA amount of HeLa cells after 1000. mu.M hydrogen peroxide treatment.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application 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 example embodiments according to the present application. 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.
The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.
As mentioned previously, conventional OG detection methods include High Performance Liquid Chromatography (HPLC) and HPLC combined with gas chromatography-mass spectrometry (GC-MS), but they both require complicated instruments and cumbersome procedures, or long incubation at high temperature for gas chromatography-mass spectrometry. Single cell gel electrophoresis is cost effective for the detection of cellular OG levels, but does not provide quantitative information. In recent years, enzymes with good specificity for OG have been used to initiate the DNA base excision repair pathway and remove OG, leaving an apurinic site that can be converted to DNA fragmentation by a cleaving enzyme. Synthetic DNA fragmentation can be measured by PCR and next generation sequencing, but these methods involve multiple enzymatic reactions and do not quantify OG levels in a particular sequence. Therefore, sensitive detection of low abundance OG levels in a particular sequence remains a significant challenge.
In view of the above, the present invention provides a method for sensitively detecting telomeric OG by using Bsu polymerase-mediated fluorescence encoding method (fig. 1). One of the major oxidation products of G is 8-oxo-7, 8-dihydroguanine (OG), and structural studies have shown that OG can adopt two conformations (cis or trans) in the active site of DNA polymerase, with the potential for dual coding. The trans conformation of OG can base pair with cytosine, while the cis form of OG can form a stable mispair with trans adenine by base pairing. We designed a 24nt oxidative damage sequence (telomere-OG), which is a DNA sequence derived from the telomere sequence. We modified an OG site at 10nt base site 5' to telomere-OG (FIG. 1). In addition, a 5' end biotin modified 14nt DNA capture probe is designed, and the probe can be hybridized with a telomere sequence to form a partial double-stranded structure. Since oxidation of G at C8 can convert the hydrogen bond acceptor (N7) into a hydrogen bond donor, a stable base pair can be formed between OG and adenine (a) (fig. 2A). However, a stable base pair cannot be formed between G and adenine (A) (FIG. 2B). Since Bsu polymerase has the greatest discrimination ability (34.1-fold) for OG and G in single nucleotide primer extension reactions among all polymerases, we selected Bsu polymerase for telomere-OG discrimination and coding. To enrich for telomere sequences from the whole genome, telomeres were isolated from genomic DNA using 5' biotinylated capture probes specific for telomeres, followed by streptavidin-coated Magnetic Beads (MB). Cy5-dATP (FIG. 1) was inserted into the capture probe at the position complementary to OG using Bsu polymerase to generate Cy 5-labeled double-stranded DNA, which was immobilized on the MB surface by the specific action of streptavidin and biotin, forming an MB-capture probe-Cy 5 complex. After magnetic separation, exonuclease iii (exoiii) was added to digest the Cy5 labeled capture probe to mononucleotides, releasing Cy5 fluorescent molecules from the MB surface. The released Cy5 fluorescent molecules can be simply counted by single molecule detection. It is worth noting that Bsu polymerase mediated fluorescence encoding enables the method to have high recognition capability, and introduction of single molecule detection greatly reduces sample consumption and improves detection sensitivity. Is tested by experimentsThe method can sensitively detect the detection rate as low as 2.45X 10-18OG of M, even 0.01% OG levels can be distinguished from complex mixtures. In addition, the method can accurately quantify OG in genomic DNA extracted from the HeLa cells treated by hydrogen peroxide, and therefore, the method has great potential in the aspects of disease-specific gene damage research and early clinical diagnosis.
Accordingly, in an exemplary embodiment of the invention, a Bsu polymerase mediated fluorescence-encoded sensor is provided, the fluorescence-encoded sensor comprising at least a DNA capture probe, a Bsu polymerase and a fluorophore modified dATP;
the DNA capture probe has a complementary region which is hybridized with a sequence to be detected, so that the DNA capture probe can be hybridized with the sequence to be detected to form a partial double-stranded structure, but the DNA capture probe does not comprise a base sequence which is complementarily paired with an OG site.
In another embodiment of the present invention, the 5' end of the DNA capture probe is modified with biotin, so that the DNA capture probe can be bound to streptavidin-coated Magnetic Beads (MB);
since Bsu polymerase has the greatest (34.1-fold) ability to discriminate OG from G in single nucleotide primer extension reactions among all polymerases, Bsu polymerase was selected for telomere-OG discrimination and encoding.
In the fluorophore modified dATP, the fluorophore is not particularly limited, and in one embodiment of the present invention, the fluorophore may be Cy5, and Cy5-dATP is embedded into the capture probe at the OG complementary position by using Bsu polymerase to generate Cy 5-labeled double-stranded DNA, which can be immobilized on the MB surface by the specific action of streptavidin and biotin to form an MB-capture probe-Cy 5 complex. Thus, the fluorescence-encoded sensor further comprises streptavidin-coated magnetic beads.
After magnetic separation, exonuclease iii (exoiii) was added to digest the Cy 5-labeled capture probe to mononucleotides, releasing Cy5 fluorescent molecules from the MB surface. The released Cy5 fluorescent molecules can be simply counted by single molecule detection. Thus, in yet another embodiment of the present invention, the fluorescence-encoded sensor further comprises exonuclease III.
Of course, the fluorescence-encoded sensor may further include a buffer and other common reagents, which can be selected and used by those skilled in the art according to routine practice, and is not limited thereto.
In yet another embodiment of the present invention, there is provided the use of the above-described fluorescence-encoded sensor for detecting 8-oxo-7, 8-dihydroguanine.
In yet another embodiment of the present invention, there is provided a method for detecting 8-oxo-7, 8-dihydroguanine, said method comprising detecting with the above-described fluorescence-encoded sensor.
Specifically, the method comprises the following steps:
s1, incubating the sample to be tested and the DNA capture probe to obtain a hybridization product;
s2, adding the hybridization product into a solution containing Bsu polymerase and fluorescent group modified dATP for continuous reaction to obtain fluorescent group labeled biotinylated double-stranded DNA;
s3, enriching the fluorescent group-labeled biotinylated double-stranded DNA obtained in the step S2 by using streptavidin-coated magnetic beads to obtain an MB-capture probe-fluorescent group complex, adding exonuclease III into the MB-capture probe-fluorescent group complex, and carrying out incubation and magnetic separation.
Wherein, in the step S1, the incubation conditions are: incubation at room temperature for 10-60 minutes (preferably 15 minutes);
in the step S2, the reaction conditions are as follows: reacting at 20-40 deg.C (preferably 30 deg.C) for 10-60 min (preferably 20 min);
in the step S3, the specific conditions of mixed enrichment are as follows; mixing at room temperature for 1-20 minutes (preferably 8 minutes);
the incubation specific conditions are as follows: the reaction is carried out at 20 to 40 deg.C (preferably 30 deg.C) for 10 to 60 minutes (preferably 15 minutes).
In another embodiment of the present invention, the method further comprises performing detection analysis on the reaction product obtained in step S3.
The detection assay includes, but is not limited to, fluorescence detection assay, single molecule detection assay, gel electrophoresis assay, and the like.
The fluorescence detection assay comprises: emission spectra in the range of 650 to 750nm were obtained at an excitation wavelength of 635nm, and data analysis was performed with an emission intensity at 670nm (maximum emission of Cy 5).
The single molecule detection assay comprises: single molecule images were obtained using a Total internal reflection fluorescence microscope (TIRF), with a 640nm laser exciting Cy5 molecules, after which Cy5 molecular counts were performed.
It should be noted that the sample to be tested may be a biological sample, and the biological sample includes blood, body fluid, tissue, cells and subcellular structures (such as telomeres) isolated from the body.
In yet another embodiment of the present invention, there is provided the use of the fluorescence-encoded sensor and/or the detection method described above in 8-oxo-7, 8-dihydroguanine-related drug screening and/or 8-oxo-7, 8-dihydroguanine analysis of biological samples.
The biological samples comprise isolated blood, body fluid, tissues, cells and subcellular structures (such as telomeres), and tests prove that the fluorescence coding sensor can sensitively detect 8-oxo-7, 8-dihydroguanine of the telomeres of the cells, so that the fluorescence coding sensor has extremely wide application value in the fields of biomedical basic research, early clinical diagnosis and the like.
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. In the following examples, nucleotide sequences using related probes and the like are shown below:
TABLE 1 oligonucleotide sequencesα
αUnderlined bold G indicates 8-oxo-7, 8-dihydroguanine (OG).
Examples
Experimental methods
Bsu polymerase mediated fluorescence encoding method for detecting OG in telomere
mu.L of 10 Xbuffer (100mM Tris-HCl, 10mM (NH)4)2SO43mM magnesium chloride, pH8.0), a concentration of oxidative damage sequence (telomere-OG) and biotin-modified capture probe were added to the reaction mixture to form a system with a total volume of 10. mu.L. The system was incubated at room temperature for 15min to obtain a hybrid product. The hybridization product was then added to a solution containing 2.5. mu.M Cy5-dATP, 0.625UBsu polymerase, and 2. mu.L 10 XNEBuffer 2(500mM sodium chloride, 100mM Tris-HCl, 100mM magnesium chloride, 1mg/mL BSA, pH7.9) in a final volume of 20. mu.L, followed by reaction at 30 ℃ for 20 minutes to obtain Cy 5-labeled biotinylated double-stranded DNA (dsDNA).
The streptavidin-coated magnetic bead solution (10mg/mL) was transferred to a 200. mu.L centrifuge tube, washed 2 times with 1 XB & W buffer (5mM Tris-HCl, 0.5mM EDTA, 1M sodium chloride, pH7.5), and the supernatant was removed. The magnetic beads were resuspended in 2 XB & W buffer (10mM Tris-HCl, 1mM EDTA, 2M sodium chloride, pH7.5) to a final concentration of 5. mu.g/. mu.L. mu.L of 5. mu.g/. mu.L MBs was mixed with 20. mu.L Cy 5-labeled biotinylated dsDNA for 8min at room temperature in a roller mixer to obtain the MB-capture probe-Cy 5 modified complex. The mixture was washed 3 times with 1 XB & W buffer (5mM Tris-HCl, 0.5mM EDTA, 1M sodium chloride, pH7.5) to remove unconjugated probes. Subsequently, the complex of MB-capture probe-Cy 5 was resuspended in a solution containing 12.5U of ExoIII, 10 XNEBuffer 1(100mM ditriphane, 100mM magnesium chloride, 1mM DTT, pH7) to a final volume of 20. mu.L, followed by incubation at 30 ℃ for 15 min. After magnetic separation, the supernatant was assayed.
2. Fluorescence detection assay
The reaction products were measured using an FLS-1000 fluorescence spectrometer (Edinburgh instruments, UK). An emission spectrum in the range of 650 to 750nm was obtained at an excitation wavelength of 635nm, and data analysis was performed with an emission intensity at 670nm (maximum emission amount of Cy 5).
3. Single molecule detection
Single molecule images were obtained using total internal reflection fluorescence microscopy (TIRF). After 500-fold dilution, TIRF imaging was performed with 10. mu.L of the reaction product. The Cy5 molecule was excited with a 640nm laser. Photons were collected using an oil-immersed 100 x objective and imaged on an EMCCD camera. For data analysis, image J software was used to select an imaged area of 600 × 600 pixels for Cy5 molecular counting. An average Cy5 count was obtained by counting 9 frames. For the unimolecular photobleaching experiments, diluted reaction mixtures were excited continuously with a 640nm laser (20mW) and real-time images were recorded at high frequency for 100ms exposure time. 200 frames were used for data analysis. All measurements were performed at room temperature.
4. Gel electrophoresis analysis
Bsu polymerase-mediated fluorescence-encoded reaction products were stained with SYBR Gold and analyzed by 20% native polyacrylamide gel electrophoresis (PAGE) in 1 XTDA-EDTA (TBE) buffer (9mM EDTA, pH 7.9). The reaction was carried out at room temperature at a constant voltage of 110V for 80min, and the gel electrophoresis image was observed by means of a Bio-Rad Chemi DocMP imaging system.
5. Detection of oxidative damage in serum samples
To investigate the feasibility of this method for real sample analysis, recovery was determined using 20 μ L fetal bovine serum 10%, 250nM capture probe, 2.5 μ MCy5-dATP, 0.0625UBsuDNA polymerase and varying concentrations of oxidative damage sequence. Recovery (R) calculation formula:
wherein C ismAnd C0Each represents the concentration of oxidative damage in the presence or absence of 10% fetal bovine serum.
6. Cell culture and preparation of genomic DNA
Cells were cultured in DMEM medium containing 10% fetal bovine serum (Cellmax, beijing, china) and 1% penicillin-streptomycin at 37 ℃ with 5% carbon dioxide. To induce oxidative damage of DNA in vivo, cells were washed with PBS buffer and treated in hydrogen peroxide incubators at different concentrations for 1 h. Genomic DNA was extracted from cells using MiniBEST Universal genomic DNA extraction kit (Dalian, China). To reduce spontaneous OG base formation during DNA preparation, antioxidants (100mM deferoxamine and 100mM butylated hydroxytoluene) were used for all reactions. According to the manufacturer's protocol, use dsDNA fragment enzyme digestion to 50-200bp, and at 95 degrees C degeneration into single chain. The amount of genomic DNA was determined using a NanoDropdrop2000c spectrophotometer (Thermo science, Wilmington, DE, USA).
Results of the experiment
1. Feasibility analysis
We performed non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis to validate the Bsu polymerase-induced fluorescent encoding process. As shown in FIG. 2C, in the presence of Bsu polymerase, a distinct new band (double-stranded DNA formed by hybridization of the capture probe to telomere-OG) was observed (lane 4), which is clearly distinguished from the pure band of telomere-OG (lane 1). The 14nt capture probe (lane 2) and the product of the control experiment (partially double-stranded DNA formed by hybridization of the capture probe to normal telomeres) indicate that Bsu polymerase can add Cy5-dATP at the exact position opposite to the OG site. We further performed fluorescence detection to validate the method. As shown in fig. 2D, a significant Cy5 fluorescence signal was detected in the presence of OG. In contrast, in the presence of normal telomeres without oxidative base damage, no significant Cy5 signal was observed, as Cy5-dATP could not be incorporated into the opposite position of G. Thus, after the ExoIII digestion reaction, the Cy5 fluorescent molecule was not released into solution (scheme 1) and no Cy5 signal was detected. In addition, we performed single molecule imaging (fig. 3). In response to the OG probe, a distinct fluorescent spot of Cy5 was observed (fig. 3B), but in response to normal telomere sequences, no fluorescent spot of Cy5 was detected (fig. 3A). Notably, Cy5 fluorescent spots exhibited a single step photobleaching (fig. 4), confirming the presence of a single molecule at each site. These results indicate that the method can be used for OG sensitivity detection.
2. Sensitivity detection
Under optimized reaction conditions, we evaluated the sensitivity of the method. As in fig. 3C, Cy5 counts increased from 0.5aM to 500nM with increasing oxidative damage concentration. In the range of 0.5aM to 5pM, Cy5 counts were linearly related to the logarithm of the oxidative damage concentration (FIG. 3D), with the correlation equation being N646.18 +33.18log10C(R20.9968) where C is the oxidative damage concentration (M) and N is Cy5 counts. By passingThe mean signal plus three times the standard deviation of the control group was calculated to determine the limit of detection to be 2.45X 10-18And M. Compared with an aptamer-based fluorescence method (3nM), the method has the advantages that the sensitivity is improved by 9 orders of magnitude, compared with an HPLC-MS method, the sensitivity is improved by 8 orders of magnitude (0.3nM), and compared with a surface enhanced Raman spectroscopy (0.1nM), the sensitivity is improved by 7 orders of magnitude.
3. Specificity analysis
To investigate the selectivity of this method, we synthesized KRAS sequences containing oxidative damage (KRAS) and random DNA sequences containing oxidative damage (random DNA) as controls. As shown in FIG. 5, in the presence of telomere-OG, a high fluorescence signal could be detected, but no significant fluorescence signal could be detected in the presence of KRAS sequence, random DNA sequence and normal telomere sequence. Since only telomeres can be captured by the telomere-specific capture probe, only OG can form stable pairings with Cy 5-dATP. In addition, we demonstrate that the method also has good selectivity for detecting oxidative damage at specific sites of telomeric DNA by measuring Cy5 signals generated by OG synthesis sequences (Table 1, DNA-1, DAN-2) at different sites of telomeric DNA. In the presence of telomere-OG, only a significant Cy5 signal was detected, but no significant Cy5 signal was detected in the presence of normal telomeres, DNA-1, DNA-2. These results indicate that the method can accurately measure OG at a specific site of telomeric DNA.
4. Detection of OG in complex systems
To investigate the ability of this method to detect OG in complex samples, we measured the level of oxidative damage in a mixture of normal telomere sequences and oxidative damage sequences. The results in fig. 6A show that the OG content measured is linearly related to the OG content of the mixture added, in the range of 0.01% to 100%. The regression equation is that Y is 1.06X-0.002 (R)20.9978) where Y is the measured OG level (%) and X is the added OG content (%). This result indicates that this method can discriminate OG levels as low as 0.01% without interference from excessive amounts of normal telomere sequences.
We further investigated the feasibility of this approach in complex biological sample analysis by testing the addition of oxidative damage assays to 10% serum. The experimentally determined recovery was 94.60-102.12% and the Relative Standard Deviation (RSD) was 0.49-1.34% (table 2), indicating a good reproducibility of the method for real sample analysis.
TABLE 2 Resuscitation study of oxidative damage in serum samples
5. Cell experiments
We also measured OG levels in telomeric DNA after hydrogen peroxide treatment. The OG level increased with increasing hydrogen peroxide concentration from 0-1000. mu.M (FIG. 6B). Indicating that hydrogen peroxide treatment can induce OG production and elevated OG levels in telomeric DNA. We examined OG levels of different types of human cell lines, such as a human cervical cancer cell line (HeLa cells), a human lung adenocarcinoma cell line (A549 cells), and a normal human liver cell line (HL-7702 cells), after treatment with 1000. mu.M hydrogen peroxide. As shown in fig. 6C, both the Cy5 count (fig. 6C) for HeLa cells and the Cy5 count (fig. 6C) for a549 cells were greater than the Cy5 count 1 (fig. 6C) for HL-7702 cells, indicating that OG levels were higher in cancer cells than in normal cells, consistent with the higher level of DNA damage in cancer patients. We further measured the change in Cy5 counts with increasing amounts of genomic DNA in 1000 μ M hydrogen peroxide treated HeLa cells. The Cy5 counts had a good linear correlation with the log of the amount of genomic DNA (0.01-100 ng) (FIG. 6D). The corresponding equation is 41.51+84.09log10X(R20.9985), where X is the number of genomic DNA of HeLa cells and N is counted as Cy 5. The detection limit is 0.0094ng, which is superior to a fluorescence method (1 mu g) based on formamidopyrimidine glycosylase and an isotope dilution method (5 mu g) based on HPLC-MS.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> university of Shandong Master
<120> Bsu polymerase-mediated fluorescence encoding sensor and application thereof in 8-oxo-7, 8-dihydroguanine detection
By using
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Claims (10)
1. A Bsu polymerase mediated fluorescence-encoded sensor comprising at least a DNA capture probe, a Bsu polymerase and a fluorophore modified dATP;
wherein the DNA capture probe has a complementary region hybridized with the sequence to be detected, so that the DNA capture probe can be hybridized with the sequence to be detected to form a partial double-stranded structure.
2. The fluorescence-encoded sensor of claim 1, wherein the 5' end of the DNA capture probe is modified with biotin.
3. The fluorescence-encoded sensor of claim 1, wherein the fluorophore is Cy5 in the fluorophore-modified dATP.
4. The fluorescence-encoded sensor of claim 1, wherein the fluorescence-encoded sensor further comprises streptavidin-coated magnetic beads and exonuclease III.
5. The fluorescence-encoded sensor of claim 1, wherein the fluorescence-encoded sensor further comprises a buffer.
6. Use of the fluorescently encoded sensor of any of claims 1 to 5 for detecting 8-oxo-7, 8-dihydroguanine.
7. A method for detecting 8-oxo-7, 8-dihydroguanine, wherein the method comprises detecting 8-oxo-7, 8-dihydroguanine using a fluorescence-coded sensor according to any one of claims 1 to 5.
8. The method of claim 7, wherein the method comprises:
s1, incubating the sample to be tested and the DNA capture probe to obtain a hybridization product;
s2, adding the hybridization product into a solution containing Bsu polymerase and fluorescent group modified dATP for continuous reaction to obtain fluorescent group labeled biotinylated double-stranded DNA;
s3, enriching the fluorescent group-labeled biotinylated double-stranded DNA obtained in the step S2 by using streptavidin-coated magnetic beads to obtain an MB-capture probe-fluorescent group compound, adding exonuclease III into the MB-capture probe-fluorescent group compound, and performing incubation and magnetic separation;
preferably, the method further comprises performing detection analysis on the reaction product obtained in step S3;
further preferably, the detection assay comprises a fluorescence detection assay, a single molecule detection assay and a gel electrophoresis assay.
9. The method of claim 8, wherein the test sample is a biological sample comprising ex vivo blood, body fluids, tissues, cells and subcellular cells.
10. Use of the fluorescence-encoded sensor and/or detection method of any of claims 1-5 in 8-oxo-7, 8-dihydroguanine-related drug screening and/or biological sample 8-oxo-7, 8-dihydroguanine detection assays.
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| CN110144384A (en) * | 2019-06-03 | 2019-08-20 | 山东师范大学 | The fluorescence chemical sensor and its detection method of a kind of test side intragranular oxidative damage and application |
| CN111154839A (en) * | 2020-01-20 | 2020-05-15 | 山东师范大学 | Fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases, detection method and application thereof |
| CN113652473A (en) * | 2021-06-30 | 2021-11-16 | 山东师范大学 | Fluorescent chemical sensor for single-molecule detection of DNA damage sites, method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110144384A (en) * | 2019-06-03 | 2019-08-20 | 山东师范大学 | The fluorescence chemical sensor and its detection method of a kind of test side intragranular oxidative damage and application |
| CN111154839A (en) * | 2020-01-20 | 2020-05-15 | 山东师范大学 | Fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases, detection method and application thereof |
| CN113652473A (en) * | 2021-06-30 | 2021-11-16 | 山东师范大学 | Fluorescent chemical sensor for single-molecule detection of DNA damage sites, method and application |
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| Title |
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| FENG TANG 等: "Location analysis of 8-oxo-7, 8-dihydroguanine in DNA by polymerase-mediated differential coding", 《THE ROYAL SOCIETY OF CHEMISTRY》, 31 December 2019 (2019-12-31), pages 4272 * |
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