CN110846385A - Biosensor for detecting and repairing glycosylase and detection method and application thereof - Google Patents
Biosensor for detecting and repairing glycosylase and detection method and application thereof Download PDFInfo
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Abstract
The invention provides a biosensor for detecting and repairing glycosylase, and a detection method and application thereof. The biosensor comprises a stem-loop DNA template and a linear primer probe; the linear primer probe is an LAMP primer probe, at least 4 primer probes are arranged, and the linear primer probe comprises two front outer inward primers, namely FIP and FOP, and two rear front outer inward primers, namely BIP and BOP. The stem region of the stem-loop DNA template is designed with a target base of the glycosylase to be repaired; the stem-loop DNA template is also provided with a region which is complementary to or has the same sequence with the primer probe sequence. The biosensor prepared by the invention actually constructs a self-guided replication system, and can be used for detecting and repairing the activity of glycosylase with zero background, high sensitivity and high specificity. Provides a simple, robust and universal platform for the biomedical research and early clinical diagnosis related to the repair enzyme.
Description
Technical Field
The invention belongs to the technical field of biological enzyme detection, and particularly relates to a biosensor for detecting and repairing glycosylase, and a detection method and application thereof.
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.
Genomic DNA consists of Watson-Crick paired heterocyclic bases for the preservation and dissemination of genetic information. Maintaining the accuracy and integrity of genomic DNA is therefore an essential prerequisite for all organisms. However, genomic DNA is often damaged by various endogenous and exogenous factors, resulting in about 10 per cell per day5Damage (e.g., modified bases, abasic sites, DNA adducts and DNA strand breaks, intra-and inter-strand cross-links, etc.). Continued DNA damage may severely induce base substitutions, insertions, deletions, and chromosomal rearrangements, leading to genomic instability, premature aging, developmental disorders, and carcinogenesis. Cells have therefore evolved a variety of protective systems to specifically repair a wide range of DNA damage, with Base Excision Repair (BER) being the most important repair mechanism that can repair a variety of DNA damage caused by oxidative, alkylation, methylation, deamination and hydrolysis reactions. DNA glycosylases are one of the most important DNA repair enzymes that initiate the critical first step of the BER pathway by cleaving the N-glycosidic bond between a damaged base and the carbon backbone of DNA. Dysregulation of DNA glycosylase is closely associated with a variety of human diseases, including neurodegenerative diseases, immunodeficiency, hypoalbuminemia, lymphoma, leukemia, dry pigmented skin disease, cocaine syndrome, and cancer (e.g., lung, breast, stomach, gall bladder, oral and colon cancers). Therefore, as a potential biomarker for clinical diagnosis and a pharmacological target for anti-cancer treatment, the development of a method capable of accurately and sensitively detecting the activity of DNA glycosylase has important significance for the research of carcinogenesis and the research and development of anti-cancer drugs.
To date, various methods have been developed for detecting DNA glycosylase activity. In principle, quantification of DNA glycosylase activity can be achieved in two modes: one by measuring the amount of damaged bases released and the other by measuring the amount of DNA product containing Abasic (AP) sites. The conventional method for detecting the activity of the DNA glycosylase is mainly based on the former mode and comprises High Performance Liquid Chromatography (HPLC), Mass Spectrometry (MS) and enzyme-linked immunosorbent assay (ELISA). However, in addition to laborious procedures, HPLC and MS often suffer from high background signals due to DNA glycosylases produced by human factors generated during sample collection and preparation. The ELISA would underestimate the actual DNA glycosylase level due to sample loss during the multi-step washing procedure. To overcome these limitations, the latter model can be used to directly detect DNA products containing AP sites, the main methods including colorimetric, electrochemical and fluorescent methods. Despite their improved performance, each approach has its own limitations in practical applications. For example: colorimetric methods require time-consuming preparation of gold nanoparticle (AuNP) probes. Electrochemical methods require cumbersome probe immobilization and complex preparation of graphene deposition electrodes. Fluorescence methods based on Quantum Dot (QD) nanosensors and DNA repair response molecular beacons can effectively detect the activity of DNA glycosylase, but they all involve complicated manipulations. While the introduction of exonuclease III (Exo III), lambda exonuclease (lambda exo) and endonuclease IV (endo IV) can be used for amplifying target signals, so that the detection sensitivity is greatly improved, but the introduction of the exonuclease III (lambda exo), the lambda exonuclease (lambda exo) and the endonuclease IV (endo IV) can not avoid the high cost of fluorophore and quencher molecule labels and the low specificity and high background caused by nonspecific nuclease digestion. Recently, a series of enzyme-based exponential amplification methods have been developed for sensitive quantitation of low abundance targets, including Polymerase Chain Reaction (PCR), branched Rolling Circle Amplification (RCA), exponential isothermal amplification (EXPAR) and Ligase Chain Reaction (LCR). PCR is a standard enzymatic DNA amplification technique based on thermocycling mediation, but involves longer assay times and precise thermocycling. Branched RCA and EXPAR are isothermal enzymatic amplification techniques, however, require multiple tool enzymes (i.e., polymerase and nickase) and are complex to operate and costly to perform. In addition, PCR, branched RCA and EXPAR are two or one primer-dependent amplification techniques, and cross-contamination due to non-specific amplification cannot be avoided. While LCR is based on the adjacent repetitive cyclic ligation of thermostable DNA ligase-mediated hybridized DNA probes, LCR product analysis is always challenged by electrophoretic separation.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a biosensor for detecting and repairing glycosylase, and a detection method and application thereof. The biosensor for detecting the repair glycosylase can be used for detecting the activity of the repair glycosylase in real time without background and label as a novel self-guided replication system. 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 invention, a biosensor for detecting a repairing glycosylase is provided, the biosensor comprises a stem-loop DNA template and a linear primer probe; the linear Primer probe is a loop-mediated isothermal amplification (LAMP) Primer probe, at least 4 Primer probes are arranged, and the linear Primer probe specifically comprises two front inner primers (FIP, Forward inner primers and FOP, Forward Outer primers) and two rear front inner primers (BIP, Backward inner primers and BOP, Backward Outer primers).
The stem region of the stem-loop DNA template is designed with a target base of the glycosylase to be repaired; the stem-loop DNA template is also provided with a region which is complementary to or has the same sequence with the primer probe sequence. The stem-loop DNA template thus serves both as a catalytic substrate for repair of glycosylases and as a template for strand displacement polymerization.
The repair glycosylase includes, but is not limited to, 8-hydroxyguanine DNA glycosylase (OGG1), alkyl adenine DNA glycosylase (AAG), formamidopyrimidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), thymine-DNA glycosylase (TDG), and the like.
The middle ring area of the stem-loop DNA template is provided with FOPc, F2c and F1 areas;
the stem region near the 5' end in the stem-loop DNA template is provided with B1, B2 and BOP regions.
The 3' end of the stem-loop DNA template is also provided with Phosphoryl (PO)4)。
The biosensor for detecting and repairing the glycosylase further comprises DNA polymerase, in particular Bst DNA polymerase.
In a second aspect of the invention, there is provided the use of the above biosensor in the detection of a repairing glycosylase.
In a third aspect of the present invention, there is provided an assay for repairing glycosylase, the assay comprising:
adding a sample to be detected into a stem-loop DNA template, and carrying out mixed incubation treatment;
and adding a linear primer probe into the solution after the incubation treatment for the incubation treatment.
The detection method further comprises detecting and analyzing the excision and amplification products by using real-time fluorescence measurement and/or gel electrophoresis analysis, thereby quantitatively detecting the repair glycosylase. SYBR Green I is used in real-time fluorescence measurement, and has high selectivity on dsDNA, so that the background can reach absolute zero signals.
In a fourth aspect of the invention, the biosensor and/or the detection method are applied to drug screening and biological sample enzyme analysis related to repair glycosylase.
The invention has the beneficial technical effects that:
(1) low background signal: based on the high precision of the repair glycosylase catalyzed target base excision repair, the high specificity of multiple primer-dependent amplifications can effectively inhibit non-specific amplification, and the high selectivity of SYBR Green I to dsDNA, and the background can reach an absolute zero signal.
(2) The sensitivity is high: based on the high efficiency of self-directed exponential replication and the absolute zero background generated by the effective inhibition of a plurality of primer-dependent amplifications on non-specific amplification, the high sensitivity of detection can be realized.
(3) The specificity is good: based on the excision repair of the damaged bases by the DNA glycosylase and the induction of self-replication by the multiple primer-dependent amplification, the non-specific amplification in the technical scheme can be efficiently inhibited, the absolute background is zero, and the high specificity of the detection can be ensured.
(4) The operation is simple: the whole reaction can be carried out in one tube under isothermal conditions, only one single type of polymerase is involved, and the output signal is detected in a label-free and real-time manner.
In conclusion, the biosensor for detecting and repairing glycosylase provided by the invention is used as a novel self-guided replication system for zero deletionBackground, no mark, real-time detection and repair of glycosylase activity. DNA glycosylases can catalyze the excision repair of damaged bases, automatically initiating self-directed replication through cyclic polymerization extension and strand displacement DNA synthesis, producing exponentially amplified dsDNA. The resulting dsDNA product can be monitored in real time without labeling using SYBR Green I as a fluorescent indicator. Based on the absolute zero background generated by the high efficiency of self-directed exponential replication and the effective inhibition of a plurality of primer-dependent amplifications on non-specific amplification, the method shows ultra-high sensitivity, and the detection limit reaches 1 multiplied by 10 in vitro-8U/. mu.L and 1 cell level in vivo. Provides a simple, robust and universal platform for biomedical research and early clinical diagnosis related to the repair enzyme, and has good practical application value.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of a self-directed replication system for the detection of repair glycosylase activity in example 1 of the present invention. The method comprises two consecutive reaction steps: (A) DNA repair-driven formation of double stem-loop DNA to initiate subsequent self-directed exponential replication, (B) initiation of self-directed exponential replication to generate large quantities of dsDNA product.
FIG. 2A is an analysis of the products of hOGG1 catalyzed 8-oxoG cleavage repair reaction under different conditions using denaturing polyacrylamide gel electrophoresis in example 1 of the present invention. Lane 1, DNA template; lane 2, in the presence of hOGG1+ DNA template; lane 3, synthetic cleavage product.
FIG. 2B is an analysis of the products of the self-directed replication reaction by denaturing polyacrylamide gel electrophoresis in example 1 of the present invention. Lane 1, synthetic BIP; lane 2, synthetic FIP; lane 3, in the presence of hgg 1; lane 4, in the absence of hgg 1; lane 5, synthetic primers FOP and BOP.
FIG. 2C shows real-time fluorescence monitoring of the amplification reaction in the presence and absence of hOGG1 in example 1 of the present invention. In this experiment, the concentration of hOGG1 was 0.25U/. mu.L.
FIG. 3A is a real-time fluorescence curve in response to various concentrations of hOGG1 in example 1 of the present invention.
FIG. 3B is a linear relationship between POI values and the logarithm of the concentration of hOGG1 in example 1 of the present invention. Error bars represent standard deviations of three independent experiments.
FIG. 4 is a real-time fluorescence curve in response to 0.1U/. mu.L hOGG1, 0.1U/. mu.L hAAG, 0.1U/. mu.L UDG, 0.1 g/. mu.L LBSA, and a control group (reaction solution only) in inventive example 1. Error bars represent standard deviations 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. 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 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 methods for detecting the activity of DNA glycosylase have the problems of time and labor waste, high background signal, low sensitivity and the like.
In view of the above, the invention utilizes the intrinsic advantages of the in vivo natural repair mechanism and loop-mediated isothermal amplification (LAMP) to construct a biosensor for detecting and repairing glycosylase for the first time, and the biosensor can be actually used as a self-guided replication system for detecting and repairing the activity of glycosylase with zero background, high sensitivity and high specificity. In a reaction assay, the entire reaction can be performed in isothermal conditions, one tube, and only one single type of polymerase is involved, and signals with a wide dynamic range can be detected in an output in a label-free, real-time manner. In addition, the protocol can be used for screening potential inhibitors, quantifying DNA glycosylase activity in a variety of cancer cells, and even detecting various DNA repair enzymes by simply changing specific damaged bases in a DNA template.
Specifically, in one embodiment of the invention, a biosensor for detecting a repair glycosylase is provided, wherein the biosensor comprises a stem-loop DNA template and a linear primer probe; the linear primer probe is an LAMP primer probe, and at least 4 primer probes are arranged, and specifically comprises two front outer inward primers (FIP and FOP) and two rear front outer inward primers (BIP and BOP).
The stem region of the stem-loop DNA template is designed with a target base of the glycosylase to be repaired; the stem-loop DNA template is also provided with a region which is complementary to or has the same sequence with the primer probe sequence. The stem-loop DNA template thus serves both as a catalytic substrate for repair of glycosylases and as a template for strand displacement polymerization.
In yet another embodiment of the present invention, the repair glycosylase includes, but is not limited to, 8-hydroxyguanine DNA glycosylase (OGG1), alkyl adenine DNA glycosylase (AAG), formamidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), thymine-DNA glycosylase (TDG), and the like.
In another embodiment of the present invention, the loop region in the stem-loop DNA template is provided with regions of FOPc, F2c, and F1;
in yet another embodiment of the present invention, the stem region near the 5' end of the stem-loop DNA template is provided with B1, B2 and BOP regions.
In another embodiment of the present invention, the stem-loop DNA template further has a phosphoryl group (PO) at the 3' -end4)。
In yet another embodiment of the present invention, when the glycosylase to be repaired is hOOG1,
the stem-loop DNA template sequence is as follows: 5' -ACT TTA TGC TTC TGT TGT GTG GAATTG TGA ACAATT TCA GTA CCC GGG GAT CCT CTA CCT GCA GGC ATG CAA GCC AAC GTC GTG ACT GGGAGG CGT TAC CCG TAA TAG CGA AGA GGC CAA CTA TAC AAC CGCOTG CAT GCC TGC AGGT-P-3' (wherein underlined O represents 8-oxoG; P represents Phosphoryl (PO)4))(SEQ ID NO.1)；
The FIP primer sequence is as follows: 5'-ACA ACG TCG TGA CTG GGA AAA CCC TTT TTG GCC TCTTCG CTA TTA C-3' (SEQ ID NO.2)
The sequence of the FOP primer is as follows: 5'-TAG TAG GTT GTA TAG TT-3' (SEQ ID NO.3)
The sequence of the BIP primer is as follows: 5'-CGA CTC TAG AGG ATC CCC GGG TAC TTT TTG TTG TGTGGA ATT GTG-3' (SEQ ID NO.4)
The BOP primer sequence is as follows: 5'-TCG TAA CTT TAT GCT TC-3' (SEQ ID NO.5)
In another embodiment of the present invention, the biosensor for detecting a repairing glycosylase further comprises a DNA polymerase, specifically a Bst DNA polymerase.
In another embodiment of the present invention, there is provided a use of the above biosensor for detecting a repairing glycosylase.
In another embodiment of the present invention, there is provided an assay for repairing glycosylase, the assay comprising:
adding a sample to be detected into a stem-loop DNA template, and carrying out mixed incubation treatment;
and adding a linear primer probe into the solution after the incubation treatment for the incubation treatment.
In another embodiment of the present invention, the incubation conditions are specifically: incubating at 30-40 ℃ for 30-60 min, preferably at 37 ℃ for 40min, to perform hOGG1 catalyzed base excision reaction;
the incubation treatment conditions are specifically: incubating at 60-70 ℃ for 30-60 min, preferably at 65 ℃ for 40min, to perform the self-directed replication reaction.
In yet another embodiment of the present invention, the detection method further comprises detecting and analyzing the excision and amplification products by real-time fluorescence measurement and/or gel electrophoresis, thereby quantitatively detecting the repaired glycosylase. SYBR Green I is used in real-time fluorescence measurement, and has high selectivity on dsDNA, so that the background can reach absolute zero signals.
In yet another embodiment of the present invention, the repair glycosylase includes, but is not limited to, 8-hydroxyguanine DNA glycosylase (OGG1), alkyl adenine DNA glycosylase (AAG), formamidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), thymine-DNA glycosylase (TDG), and the like.
In another embodiment of the present invention, the biosensor and/or the detection method are used for drug screening and enzyme analysis of biological samples related to repair glycosylase.
The drugs related to repairing glycosylase comprise but are not limited to a repairing glycosylase inhibitor and a repairing glycosylase activator;
the biological samples comprise biological cells such as cancer cells and the like, and tests prove that the biosensor provided by the invention has better analysis capability on real complex biological samples, can be used for quantitatively detecting the activity of repairing glycosylase (such as hOOG1), and has ultrahigh sensitivity, and the detection limit can reach the level of 1 cell in vivo. Therefore, the method has great application potential in the fields of biomedical basic research, clinical diagnosis and the like.
The invention relates to a method for carrying out ultrasensitive detection on activity of repairing glycosylase based on a zero-background and unmarked self-guided replication system. The method mainly comprises two continuous reaction steps: (A) DNA repair-driven formation of double stem-loop DNA, initiating self-directed exponential replication, (B) initiation of self-directed replication, producing large quantities of dsDNA product.
In the first step, exemplified by human 8-oxoguanine DNA glycosylase (hOGG1), when hOGG1 is present, it will specifically recognize and excise 8-oxoG from the mismatched 8-oxoG/C base pair by cleaving the N-glycosidic bond between the sugar and the damaged base, resulting in an apurinic/Apyrimidinic (AP) site. The AP site was then efficiently excised with the aid of a human apurinic/apyrimidinic endonuclease (APE1), resulting in a single nucleotide gap. As a result, the hairpin structure of the DNA template is opened, forming a linear DNA sequence.
In the second step, two pairs of primer probes (i.e., two front inner and outer primers (FIP and FOP) and two rear inner and outer primers (BIP and BOP)) and Bst DNA polymerase were added, and FIP hybridized with F2c in the unfolded DNA template to initiate polymerization extension. Meanwhile, FOP (shorter than the sequence of FIP and lower in concentration) can hybridize with FOPc in the unfolded DNA template to initiate strand displacement DNA synthesis (SDS), generate dsdna (i) and simultaneously release single-stranded DNA (ssdna) (which can form a stem-loop structure at 5' end by hybridization between F1 and F1c and has F2 on its stem-loop). Similarly, BIP and BOP can hybridize side-by-side with B2c and BOPc in dsdna (i) above, successfully initiating polymerization extension and SDS, generating dsdna (ii) and simultaneously releasing a ssDNA that can form a double-stem loop (which forms loop structures containing F2c and B2 at the 5 'and 3' ends by hybridization of B1 and B1c, respectively, F1 and F1c, respectively). Notably, once the double-stem loop DNA is formed, autonomous exponential replication under isothermal conditions begins. The resulting double-stem-loop DNA (I) can first initiate self-initiated polymerization extension at the 3' end to form a stem-loop DNA (loop F2 c). FIP will then hybridize to F2c on the loop to prime SDS, producing a dsDNA intermediate. The dsDNA intermediate will continue to initiate self-initiated polymerization extension and SDS, producing dsDNA (i) containing the B2c stem loop, while releasing the double stem loop dna (ii) containing F2 and B2 c. Importantly, BIP can hybridize with B2c on the loop of dsDNA (I) formed above, repeatedly initiating SDS and self-priming polymerization, thereby initiating cyclic amplification (I) to produce large amounts of stem-loop DNAs and dsDNAs intermediates containing different fragment sizes. Similarly, FIP can hybridize to F2c on loops formed in dsDNA (II) to initiate cyclic amplification (II). Through multiple rounds of self-induced replication reactions, the final result is exponential amplification of DNA and the production of large quantities of dsDNA product. The obtained dsDNA product can be used as dye to carry out real-time quantitative detection on the activity of DNA glycosylase by using SYBR Green I.
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
DNA repair driven self-directed exponential replication. First, all the synthesized oligonucleotides were dissolved in 1 XTTris-EDTA buffer (10mM Tris, 1mM EDTA, pH 8.0) to prepare stock solutions. Next, the DNA template is placed in hybridization buffer (10mM Tris-HCl, 1.5mM MgCl)2pH 8.0) at 95 degrees celsius for 5 minutes, and then slowly cooled to room temperature to form a hairpin structure. Third, 0.5. mu.l of the prepared DNA template (10. mu.M) was added to the cleavage reaction solution (10. mu.L) containing various concentrations of hOGG1, 2. mu.l of 10 XNEB buffer 2 and 2. mu.l of BSA (10X), and incubated at 37 ℃ for 40 minutes to perform a hOGG 1-catalyzed base cleavage reaction. In the fourth step, an amplification reaction solution (25. mu.L) containing 6U of Bst DNA polymerase, 250. mu.M of dNTPs, 500. mu.M of Forward Internal Primer (FIP), 500. mu.M of Backward Internal Primer (BIP), 125. mu.M of forward external primer (FOP), 125. mu.M of backward external primer (BOP) and 3.5. mu.L of 10 × ThermoPol was added to the above reaction solution and incubated at 65 ℃ for 40 minutes to perform a self-directed replication reaction.
Real-time fluorescence measurement and gel electrophoresis analysis: real-time fluorescence was monitored by the BIO-RAD CFX Connect TM real-time system (Hercules, CA, U.S.A.) and fluorescence intensity was measured simultaneously at 30 second intervals. SYBR Green I was used as a fluorescent indicator. For analysis of the cleavage and amplification products, 12% denaturing polyacrylamide gel electrophoresis (PAGE) analysis was performed in 1 XTBE buffer (9 mmol per liter Tris-HCl, 9 mmol per liter boric acid, 0.2 mmol per liter EDTA, pH 7.9) at room temperature at a constant voltage of 110V. After 50 minutes of electrophoresis, the gel was stained with a silver staining kit and analyzed by imaging with a ChemiDoc MP imaging system (Hercules, California, u.s.a).
Experimental principle (as in fig. 1):
we designed one stem-loop DNA template and four linear primer probes (i.e. two front plus-in primers (FIP and FOP) and two back plus-out primers (BIP and BOP)). Having 3' -Phosphoryl (PO)4) The terminal stem-loop DNA template serves both as a catalytic substrate for hOGG1 and as a template for strand displacement polymerization. Four Linear primer probes (i.e., FIP, BIP, FOP and BOP)Can effectively mediate self-directed exponential replication. As shown in FIG. 1A, in the presence of hOGG1, damaged 8-oxoG in the DNA template can be specifically recognized and efficiently cleaved by the BER mechanism, thereby creating a single nucleotide gap and simultaneously unfolding the loop structure of the DNA template. With the addition of two pairs of primers and Bst DNA polymerase, FIP will first hybridize to F2c in the opened DNA template to induce a polymerization reaction. Meanwhile, FOP can hybridize with FOPc in the opened DNA template to initiate strand displacement DNA synthesis (SDS), generate dsDNA (I), and release DNA containing one stem loop at the same time. Similarly, BIP and BOP can hybridize sequentially to B2c and BOPc in stem-loop DNA, successfully initiating polymerization extension and SDS, generating dsDNA (II) and simultaneously releasing a formable double-stem-loop DNA. Once double-stem-loop DNA is formed, self-directed exponential replication at constant temperature begins. As shown in FIG. 1B, the resulting double stem-loop DNA (I) can initiate self-initiated polymerization extension at the 3' end to form a stem-loop DNA containing F2c in the loop. FIP will then hybridize to F2c in the loop to prime SDS, producing a dsDNA intermediate. This intermediate will then initiate self-initiated polymerization extension and SDS, producing a single-stem-loop DNA (I), and simultaneously release a double-stem-loop DNA (II) (stem-loops containing F2 and B2c, respectively). Importantly, BIP can hybridize to B2c on the single-stem loop DNA (I) loop, repeatedly priming SDS to initiate cycle amplification (I), thereby producing large amounts of stem-loop DNA and dsDNA intermediates. Meanwhile, the released double-stem-loop DNA (II) may sequentially initiate self-priming polymerization and SDS reaction to form a single-stem-loop DNA having B2c in the loop. BIP can then hybridize to B2c in the loop to prime SDS, producing a dsDNA intermediate. The dsDNA intermediate will then initiate self-initiated polymerization extension and SDS reaction to produce a dsDNA (ii) (stem loops containing F2c and B2, respectively). FIP can hybridize to F2c on loops in dsDNA (II) to initiate cyclic amplification (II) to yield large amounts of stem-loop DNA and dsDNA intermediates. At the same time FIP can hybridize with F2c on the loop in the newly formed double-stem loop (I), automatically initiating a new cycle of polymerization extension, SDS and double-stem loop DNA generation. Eventually, the persistence of cyclic self-directed exponential replication will result in exponential amplification of DNA and the generation of large amounts of dsDNA product. The obtained dsDNA product can be used for detecting the activity of DNA glycosylase by using SYBR Green I as an indicatorAnd (4) carrying out quantitative detection.
(1) Experimental verification of the principle:
to verify whether hOGG1 could cleave 8-oxoG to induce unfolding of the loop structure in the DNA template, we analyzed the cleavage products using 12% denaturing PAGE and silver staining kit as a fluorescence indicator. As shown in FIG. 2A, in the presence of hOGG1 and DNA template (FIG. 2A, lane 2), three characteristic bands, 148, 132 and 15nt respectively, were observed, with sizes just equal to DNA template (148nt, FIG. 2A, lane 1), longer cleavage products (132nt, FIG. 2A, lane 3) and shorter cleavage products (15nt), indicating that hOGG1 can effectively cleave the damaged base 8-oxoG by BER mechanism and produce a single nucleotide gap, resulting in subsequent cleavage of the DNA template to produce two DNA fragments (i.e., longer cleavage product (132nt) and shorter cleavage product (15 nt)). In contrast, in the presence of only the DNA template (FIG. 2A, lane 1), only one band of the original DNA template (148nt) was observed, indicating that no excision reaction occurred. The amplification products were further analyzed by adding two pairs of primers (i.e., FIP and FOP, BIP and BOP). As shown in fig. 2B, in the presence of hgg 1, multiple bands of ladder pattern (fig. 2B, lane 3) were detected in addition to the original band FIP (46nt, fig. 2B, lane 2), BIP (45nt, fig. 2B, lane 1), FOP (17nt, fig. 2B, lane 5) and BOP (17nt, fig. 2B, lane 5), indicating that homing index replication was initiated and produced large amounts of stem-loop DNA and dsDNA intermediates. Furthermore, in the absence of hOGG1, no characteristic ladder-like bands were observed (FIG. 2B, lane 4), indicating that no amplification reaction occurred. To further validate the feasibility of this protocol, we performed real-time fluorescence monitoring of the entire amplification reaction using SYBR Green I as a fluorescence indicator. As shown in fig. 2C, the real-time fluorescence signal increased in a sigmoidal manner in the presence of chogg 1 (fig. 2C), indicating that chogg 1 catalyzes excision repair of 8-oxoG that can successfully induce DNA template hairpin unfolding and subsequent self-directed replication. However, in the absence of hOGG1, only a signal close to zero background was observed even at long reaction times of 120 minutes (FIG. 2C).
(2) Sensitivity test:
to investigate the detection sensitivity of the protocol, IThe fluorescence intensity was measured at various concentrations of hOGG1 under optimal conditions. As shown in FIG. 3A, as the concentration of hOGG1 was varied from 1X 10-8Increasing to 0.25U/μ L, the real-time fluorescence signal gradually increased in a sigmoidal manner as the unfolded DNA template switched from single-stranded to partially double-stranded DNA. The inflection Point (POI) is defined as the time corresponding to the maximum slope in the sigmoidal amplification curve with which we quantified the activity of chogg 1. As shown in FIG. 3B, at 1.0X 10-8A good linear relationship between POI values and log values of chogg 1 concentration over a large dynamic range of 7 orders of magnitude to 0.25U/μ L (fig. 3B inset). The corresponding equation is POI 3.5+10.8log10C, correlation coefficient 0.9913, where C is the concentration of hcogg 1. The detection limit was finally determined to be 1.0X 10-8U/μL。
(3) Specific experiments:
to investigate the specificity of this protocol, we used human alkyl adenine DNA glycosylase (hAAG), Uracil DNA Glycosylase (UDG) and Bovine Serum Albumin (BSA) as negative controls. As shown in fig. 4, in the presence of hOGG1, the real-time fluorescence signal increased in an S-shape. In contrast, in the presence of hAAG, UDG, BSA and the reaction solution only control, only a near-zero background signal was observed. The results show that the method can well distinguish hOGG1 from other interfering proteins including DNA glycosylase family members, thereby proving that the technical scheme has good specificity.
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
<110> university of Shandong Master
<120> biosensor for detecting and repairing glycosylase, and detection method and application thereof
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actttatgct tctgttgtgt ggaattgtga acaatttcag tacccgggga tcctctacct 60
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ctatacaacc gcotgcatgc ctgcaggt 148
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acaacgtcgt gactgggaaa accctttttg gcctcttcgc tattac 46
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Claims (10)
1. A biosensor for detecting and repairing glycosylase is characterized by comprising a stem-loop DNA template and a linear primer probe; the linear primer probes are LAMP primer probes, at least 4 primer probes are arranged, and the linear primer probes comprise two front positive outer inward primers, namely FIP and FOP, and two rear positive outer inward primers, namely BIP and BOP;
the stem region of the stem-loop DNA template is designed with a target base of the glycosylase to be repaired; the stem-loop DNA template is also provided with a region which is complementary to or has the same sequence with the primer probe sequence.
2. The biosensor of claim 1, wherein the repair glycosylase comprises 8-hydroxyguanine DNA glycosylase, alkyl adenine DNA glycosylase, formamidopyrimidine DNA glycosylase, uracil-DNA glycosylase, and thymine-DNA glycosylase.
3. The biosensor in accordance with claim 1, wherein the loop region in the stem-loop DNA template is provided with regions of FOPc, F2c, and F1;
the stem region near the 5' end in the stem-loop DNA template is provided with B1, B2 and BOP regions.
4. The biosensor in accordance with claim 1, wherein when the repair glycosylase to be detected is hOOG1,
the stem-loop DNA template sequence is shown in SEQ ID NO. 1;
the FIP primer sequence is shown as SEQ ID NO. 2;
the sequence of the FOP primer is shown as SEQ ID NO. 3;
the sequence of the BIP primer is shown as SEQ ID NO. 4;
the BOP primer sequence is shown in SEQ ID NO. 5.
5. The biosensor of claim 1, wherein the biosensor for detecting a repair glycosylase further comprises a DNA polymerase, preferably Bst DNA polymerase.
6. Use of the biosensor of any one of claims 1-5 for detecting a repairing glycosylase.
7. An assay for repairing a glycosylase, the assay comprising:
adding a sample to be detected into a stem-loop DNA template, and carrying out mixed incubation treatment;
and adding a linear primer probe into the solution after the incubation treatment for the incubation treatment.
8. The detection method according to claim 7, wherein the incubation treatment conditions are in particular: incubating for 30-60 min at 30-40 ℃;
the incubation treatment conditions are specifically: incubating for 30-60 min at 60-70 ℃.
9. The detection method of claim 7, further comprising detecting and analyzing the cleavage and amplification products using real-time fluorescence measurement and/or gel electrophoresis.
10. Use of the biosensor of any one of claims 1-5 and/or the detection method of claim 7 or 8 for repairing glycosylase-related drug screening, enzyme analysis of biological samples;
the medicines related to the repair glycosylase comprise a repair glycosylase inhibitor and a repair glycosylase activator;
the biological sample comprises cells of an organism.
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| CN109628556A (en) * | 2018-11-27 | 2019-04-16 | 山东师范大学 | The active method of cycle signal amplification detection people's 8- hydroxy guanine DNA glycosylase mediated based on autocatalytic replication |
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Non-Patent Citations (2)
| Title |
|---|
| L. WANG等: "Construction of a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background", 《CHEM. SCI.》 * |
| 逯莹莹: "自导向复制系统检测糖基化酶的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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