CN109988822B - Sensor and method for detecting hAAG through controllable autocatalysis cleavage mediated fluorescence recovery - Google Patents
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Abstract
The present disclosure provides a sensor and method for controllable autocatalytic cleavage mediated fluorescence recovery detection of hAAG, the sensor comprising hairpin probe 1, hairpin probe 2, signaling probe, APE1 and T7 exonuclease; hairpin probe 1 and hairpin probe 2 are DNA with a stem-loop structure with a protruding 5 '-end, the 5-end stem of hairpin probe 1 is modified with 2' -deoxyinosine which can be recognized by hAAG, hairpin probe 1 contains trigger probe 1, and trigger probe 1 contains a first DNA sequence which can be complementary with a second DNA sequence; the hairpin probe 2 consists of a trigger probe 2 and a second DNA sequence, wherein the trigger probe 2 contains a third DNA sequence which can be complementary with the signal probe; the signal probe is single-stranded DNA, and the two ends of the signal probe are respectively modified with fluorophores and quenchers. The detection method using the sensor can efficiently and sensitively detect the activity of hAAG.
Description
Technical Field
The present disclosure pertains to biological analysis technology, and relates to a sensor and method for detecting hAAG by controllable autocatalytic cleavage-mediated fluorescence recovery.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Watson-Crick base pairing DNA double helix structure is an important genetic code in organisms. In reality, since heterocyclic bases (i.e., A, T, G and C) contain a large number of nucleophilic or oxidative active sites, they readily react with electrophilic or reactive oxygen species generated extracellularly and endogenously to produce a large number of DNA alkylation and oxidative damage (e.g., 3-alkyladenine, 7-alkylguanine, 1-N) 2 Ethylene guanine, 1-N 6 Vinyl adenine, hypoxanthine, etc.). Recent studies have shown that alkylation and oxidative damage can inhibit DNA replication or create DNA mutations by blocking the polymerase, ultimately leading to the occurrence of cancer. To date, in human cells, human alkyladenine DNA glycosylationThe enzyme (hAAG) is the only glycosylase capable of specifically recognizing and cleaving a wide variety of DNA alkylating and oxidative damage, and its abnormal function will lead to failure of the mechanism of repair of the alkylated DNA damage, thereby causing a wide variety of human diseases (e.g. chronic inflammation, crohn's disease and wilson's disease) and cancers (e.g. colon cancer, liver cancer, lung cancer, cervical cancer, glioblastoma, lymphoma, melanoma and leukemia).
To the best of the inventors' knowledge, the only methods developed so far for hAAG assays are based on radioactive phosphorus (gamma-) 32 P) gel electrophoresis of labeled DNA substrates, immunoblotting assays mediated by horseradish peroxidase-conjugated immunoglobulins, and single molecule counting methods mediated by repair reaction molecular beacons. However, as studied by the inventors of the present disclosure, these methods have drawbacks of radioactive contamination, time-consuming and labor-consuming operations, strict reaction conditions, low accuracy, and the need for expensive antibodies and precise instruments.
Disclosure of Invention
In order to solve the deficiencies of the prior art, it is an object of the present disclosure to provide a sensor and method for detecting hAAG by controllable autocatalytic cleavage-mediated fluorescence recovery, which can be used to efficiently and sensitively detect the activity of human alkyl adenine DNA glycosylase (hAAG).
In order to achieve the above object, the technical scheme of the present disclosure is as follows:
in one aspect, a sensor for detection of hAAG based on controlled autocatalytic cleavage-mediated fluorescence recovery, comprising hairpin probe 1, hairpin probe 2, signaling probe, human apurinic/apyrimidinic endonuclease (APE 1), and T7 exonuclease;
the signal probe is single-stranded DNA, and the two ends of the signal probe are respectively modified with fluorophores and quenchers.
Enzyme-mediated cyclic signal amplification (CESA) is a nuclease-based, introducing a target that can cause the target-dependent nuclease to cyclically cleave the signal probe to output amplified signals, and the present disclosure selectively utilizes T7exonuclease to construct new CESA systems, i.e., based on controllable autocatalytic cleavage-mediated fluorescence recovery, to achieve simple, highly sensitive and highly specific detection of hAAG activity. T7Exonuclease (T7 Exonecut) is a unique sequence independent nuclease that selectively catalyzes the conversion of a single nucleotide from 5' -phosphoryl (PO 4 ) Or the 5' -hydroxyl (OH) end or nicks of double-stranded DNA (dsDNA), but does not act on single-stranded DNA (ssDNA)). In addition, T7 exonucleases can degrade RNA or DNA on the RNA/DNA hybrid duplex in the 5 'to 3' direction, but cannot degrade double-stranded or single-stranded RNA. More importantly, T7 exonucleases can distinguish single base mismatches at high resolution.
Hairpin probe 1 (HP 1) is specifically cleaved at the compromised 2' -deoxyinosine site when in the presence of hAAG, resulting in the expansion of the hairpin structure to produce a DNA duplex. Trigger probe 1 (trigger 1) constructed in a DNA duplex will hybridize to hairpin probe 2 (HP 2) moiety by a cohesive end-mediated strand displacement reaction (tmdr) to induce T7exonuclease to catalyze the first cycle cleavage of HP2 to release trigger probe 2 (trigger 2). The third DNA sequence of trigger probe 2 (trigger 2) is partially complementary to the signaling probe, thereby inducing a second circular cleavage of the signaling probe. By a two-step cyclic autocatalytic cleavage process, a large number of fluorescent molecules are released from the fluorophore-quencher Fluorescence Resonance Energy Transfer (FRET) pair, ultimately producing a significantly enhanced fluorescent signal for detection of hAAG activity, thereby enabling simple, rapid and sensitive detection.
In another aspect, a method for detecting hAAG based on controlled autocatalytic cleavage-mediated fluorescence recovery provides the above sensor; when hAAG exists, hAAG and APE1 specifically recognize and cleave hairpin probe 1 at the 2' -deoxyinosine site of hairpin probe 1, so that hairpin probe 1 expands hairpin structure, dsDNA duplex I with protruding 5' -OH and 5' -dRP ends is generated, protruding 5' -dRP ends in dsDNA duplex I are hybridized with a second DNA sequence of hairpin probe 2 through a strand displacement reaction mediated by sticky ends, a first DNA sequence of trigger probe 1 is hybridized with a signal probe to form dsDNA duplex II with recessed 5' -OH ends, dsDNA duplex II is used as a substrate of T7exonuclease, recessed 5' -OH ends in hairpin probe 2 are specifically hydrolyzed under the action of T7exonuclease, trigger probe 2 is released while trigger probe 2 in hairpin probe 2 is released, released trigger probe 1 is hybridized with excessive hairpin probe 2, T7exonuclease is induced to catalyze the first cycle cleavage of hairpin probe HP2 to release a plurality of trigger probes 2, a third DNA sequence of trigger probe 2 is hybridized with a signal probe to form dsDNA duplex II with recessed 5' -OH ends, dsDNA duplex III is formed as a substrate of T7exonuclease, and thus signal probe III is released from the second exonuclease, and signal III is released from the probe 2 by the exonuclease, thereby the signal III is continuously cleaved with the probe 2, and the signal III is released.
In the absence of hAAG, the 2' -deoxyinosine damage site in hairpin probe HP1 cannot be recognized, and hairpin probe HP1 is not cleaved by APE 1. Hairpin probes HP1 and HP2 with protruding 5' -OH ends therefore block the T7 exonuclease-assisted autocatalytic recycling cleavage process and fail to generate enhanced fluorescent signals.
In a third aspect, a kit for detecting hAAG comprises the above sensor and a buffer solution.
The beneficial effects of the present disclosure are:
1. the present disclosure is based on controllable self-controlThe catalytic cleavage mediated fluorescence recovery is used for detecting the activity of the human alkyl adenine DNA glycosylase with high sensitivity and high specificity, realizes the two-step amplification of fluorescent signals, has ultrahigh sensitivity and higher resolution, and has the detection limit of 4.9x10 -6 The detection range of the sensitivity is 1×10 per microliter -5 4 orders of magnitude per microliter of 0.1 units, the present disclosure can therefore efficiently and sensitively detect the activity of human alkyl adenine DNA glycosylase.
2. The present disclosure utilizes the high accuracy of DNA repair systems and the high resolution of single base mismatches by T7 exonucleases, only hAAG can specifically recognize and cleave hairpin substrates at the 2' -deoxyinosine site, thereby inducing T7 exonuclease-assisted autocatalytic recycling fluorescent signal amplification, with higher signal-to-noise ratios guaranteeing the high specificity of the present disclosure.
3. The method does not need complicated washing, transferring and separating steps, does not involve any thermal cycle, and greatly simplifies the detection procedure.
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The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate and explain the exemplary embodiments of the disclosure and together with the description serve to explain the disclosure, and do not constitute an undue limitation on the disclosure.
FIG. 1 is a schematic diagram of embodiment 1 of the present disclosure;
FIG. 2 is a schematic representation of the principle verification of example 1, A is an analytical plot of the cleavage reaction product of hAAG-guided hairpin substrate by non-denaturing polyacrylamide gel electrophoresis under different experimental conditions, lane M is a DNA marker, lane 1 is the cleavage product of synthetic hairpin probe HP1, lane 2 is the synthetic trigger probe 1, lane 3 is the synthetic hairpin probe HP1, lane 4 is the reaction product in the presence of APE1+HP1, lane 5 is the reaction product in the presence of hAAG+APE1+HP1, B is an analytical plot of the cleavage reaction product of T7 exonuclease-assisted autocatalysis by non-denaturing polyacrylamide gel electrophoresis, lane M is a DNA marker, lane 1 is the reaction product in the absence of hAAG, lane 2 is the cleavage product of synthetic trigger probe 2, lane 3 is the reaction product in the presence of hAAG, the concentrations of hAAG and APE1 are 0.1 units per microliter and 0.3 units per microliter, respectively, using BR SYld as fluorescent indicator;
FIG. 3 is a plot of the sensitivity profiles of example 1, A being the concentration of the various components (0, 1X 10 -5 U/μL、5×10 -5 U/μL、2.5×10 -4 U/μL、5×10 -4 U/μL、2.5×10 -3 U/μL、5×10 -3 U/μL、2.5×10 -2 U/μL、1×10 -1 U/. Mu.L), B is the fluorescence intensity of hAAG with the concentration of hAAG from 1X 10 -5 The curve of U/. Mu.L to 0.1U/. Mu.L is shown in the inset of FIG. B at 1X 10 -5 In the range of U/. Mu.L to 0.1U/. Mu.L, the linear relationship between fluorescence intensity and logarithm of hAAG concentration, error bars represent standard deviation of three independent experiments.
FIG. 4 is a graph representing the characterization of the specificity of example 1, a bar graph of the change in fluorescence intensity in the presence of control (reaction buffer alone), 0.1g/L BSA,0.1g/L IgG, 0.1U/. Mu.LhOGG 1 and 0.1U/. Mu.LhAAG, error bars representing the standard deviation of three independent experiments.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present 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 disclosure 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 in accordance with the present disclosure. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The present disclosure provides a sensor and method for detecting hAAG by controllable autocatalytic cleavage-mediated fluorescence recovery, which can be used for efficiently and sensitively detecting the activity of human alkyl adenine DNA glycosylase (hAAG).
In one exemplary embodiment of the present disclosure, a sensor for detection of hAAG based on controlled autocatalytic cleavage-mediated fluorescence recovery is provided, comprising hairpin probe 1, hairpin probe 2, signaling probe, human apurinic/apyrimidinic endonuclease (APE 1), and T7 exonuclease;
the signal probe is single-stranded DNA, and the two ends of the signal probe are respectively modified with fluorophores and quenchers.
Hairpin probe 1 (HP 1) is specifically cleaved at the compromised 2' -deoxyinosine site when in the presence of hAAG, resulting in the expansion of the hairpin structure to produce a DNA duplex. Trigger probe 1 (trigger 1) constructed in a DNA duplex will hybridize to hairpin probe 2 (HP 2) moiety by a cohesive end-mediated strand displacement reaction (tmdr) to induce T7exonuclease to catalyze the first cycle cleavage of HP2 to release trigger probe 2 (trigger 2). The third DNA sequence of trigger probe 2 (trigger 2) is partially complementary to the signaling probe, thereby inducing a second circular cleavage of the signaling probe. By a two-step cyclic autocatalytic cleavage process, a large number of fluorescent molecules are released from the fluorophore-quencher Fluorescence Resonance Energy Transfer (FRET) pair, ultimately producing a significantly enhanced fluorescent signal for detection of hAAG activity. Thus realizing simple, rapid and sensitive detection.
In one or more embodiments of this embodiment, the fluorophore and quencher are modified at the 5 'end and 3' end, respectively, of the signaling probe.
In one or more embodiments of this embodiment, the fluorophore is FAM and the quencher is BHQ1.
In one or more examples of this embodiment, the sequence of hairpin probe 1 is: 5' -GTA GTG AGG TAG GTT GTA TIG TTG GGT TGA ACT ATA CAA CCT ACC-3 '(wherein the underlined base I is 2' -deoxyinosine)
The sequence of hairpin probe 2 is: 5'-TGT ATA GTT CAA CCC GGG ACC TAA GAG CAT TCT ACA CCT CTT AGG TCC CTG C-3'
The sequence of the signal probe is as follows: 5'-AAG AGG TGT A-3'.
In another embodiment of the present disclosure, a method for detecting hAAG based on controllable autocatalytic cleavage-mediated fluorescence recovery is provided, providing a sensor as described above; when hAAG exists, hAAG and APE1 specifically recognize and cleave hairpin probe 1 at the 2' -deoxyinosine site of hairpin probe 1, so that hairpin probe 1 expands hairpin structure, dsDNA duplex I with protruding 5' -OH and 5' -dRP ends is generated, protruding 5' -dRP ends in dsDNA duplex I are hybridized with a second DNA sequence of hairpin probe 2 through a strand displacement reaction mediated by sticky ends, a first DNA sequence of trigger probe 1 is hybridized with a signal probe to form dsDNA duplex II with recessed 5' -OH ends, dsDNA duplex II is used as a substrate of T7exonuclease, recessed 5' -OH ends in hairpin probe 2 are specifically hydrolyzed under the action of T7exonuclease, trigger probe 2 is released while trigger probe 2 in hairpin probe 2 is released, released trigger probe 1 is hybridized with excessive hairpin probe 2, T7exonuclease is induced to catalyze the first cycle cleavage of hairpin probe HP2 to release a plurality of trigger probes 2, a third DNA sequence of trigger probe 2 is hybridized with a signal probe to form dsDNA duplex II with recessed 5' -OH ends, dsDNA duplex III is formed as a substrate of T7exonuclease, and thus signal probe III is released from the second exonuclease, and signal III is released from the probe 2 by the exonuclease, thereby the signal III is continuously cleaved with the probe 2, and the signal III is released.
In the absence of hAAG, the 2' -deoxyinosine damage site in hairpin probe HP1 cannot be recognized, and hairpin probe HP1 is not cleaved by APE 1. Hairpin probes HP1 and HP2 with protruding 5' -OH ends therefore block the T7 exonuclease-assisted autocatalytic recycling cleavage process and fail to generate enhanced fluorescent signals.
In one or more embodiments of this embodiment, the steps are as follows:
(1) Hairpin probe 1 was added to a first solution containing hAAG, 10 XThermopol reaction buffer, APE1, 10 XNEBuffer 4 and incubated with heating;
(2) Adding the material obtained in the step (1) into a second solution containing hairpin probes 2, signal probes, T7exonuclease and 10 XNEBuffer 4 for incubation;
(3) And (3) performing fluorescence detection on the incubated material in the step (2).
In this series of examples, the incubation conditions in step (1): the temperature is 37 plus or minus 0.5 ℃ and the time is 60-90 min.
In this series of examples, the concentration of APE1 in the first solution of step (1) is 0.3 units per microliter; each 20. Mu.l of the first solution contained 2. Mu.l of 10 XThermopol reaction buffer and 2. Mu.l of 10 XNEBuffer 4.
In this series of examples, in step (1), the volume ratio of the solution of hairpin probe 1 to the first solution is 1:9.5-10.5, and the concentration of hairpin probe 1 in the solution of hairpin probe 1 is 1. Mu. Mol/liter.
In this series of examples, the incubation conditions in step (2): the temperature is 25+/-0.5 ℃ and the time is 50-60 min.
In this series of examples, the concentration of hairpin probe 2 in the second solution of step (2) was 250 nanomoles per liter, the concentration of signaling probe was 700 nanomoles per liter, and the concentration of T7exonuclease was 15 units per 20 microliters, containing 2 microliters of 10 XNEBuffer 4 per 20 microliters of the second solution.
In the series of embodiments, in the step (2), the volume ratio of the material in the step (1) to the second solution is 1:4.5-5.5.
In this series of examples, in step (3), the detection excitation wavelength was 491nm and the emission wavelength was 520nm.
In order to incubate hairpin probes 1,2 into a stem-loop structure, in one or more examples of this embodiment, single-stranded hairpin probe 1 and/or single-stranded hairpin probe 2 are diluted with hybridization buffer, heated to 95 ℃ for incubation for 5min, and then cooled to room temperature, the single-stranded hairpin probe 1 and/or single-stranded hairpin probe 2 forming a stem-loop structure.
In a third embodiment of the present disclosure, a kit for detecting hAAG is provided, comprising the above-described sensor and a buffer solution.
The present disclosure contemplates two hairpin probes (i.e., HP1 and HP 2), both having prominent 5' -OH ends, in order to effectively prevent digestion by T7 exonuclease. In hairpin probe HP1, 2' -deoxyinosine (I) is modified at a site 3 bases from the loop on the stem, and this lesion site can be recognized by hAAG. The signaling probe is a DNA sequence comprising 10nt, a Fluorophore (FAM) and a quencher (BHQ 1) modified at the 5 'and 3' ends, respectively, wherein fluorescence of FAM is quenched by BHQ1 by fluorescence energy resonance transfer (FRET). T7exonuclease is the "core" of the CESA system, responsible for the operation of autocatalytic cycle signal amplification. The present disclosure generally includes two sequential reaction steps: (1) Specifically cleaving the 2' -deoxyinosine site on hairpin probe HP1 in the presence of hAAG and APE1, (2) T7exonuclease assisted autocatalytic cycle signal amplification. In the presence of hAAG, it can specifically recognize and cleave hairpin probe HP1 at the 2' -deoxyinosine site and unfold the hairpin structure, while producing dsDNA duplex (I) with protruding 5' -OH and 5' -dRP (deoxyribose phosphate) ends. The protruding 5'-dRP end in dsDNA duplex (I) (i.e., trigger probe 1) will hybridize to the hairpin probe HP2 stem portion by a cohesive end-mediated strand displacement reaction (tmmdr) to form a dsDNA duplex (II) with a recessed 5' -OH end. This newly formed dsDNA duplex (II) can serve as a substrate for T7exonuclease, and then hydrolyze specifically from the recessed 5' -OH end in HP2, releasing trigger probe 1 and simultaneously releasing trigger probe 2 in HP 2. Since the signaling probe is complementary to a partial region of trigger probe 2, released trigger probe 2 will hybridize to the signaling probe to form a dsDNA duplex (III) with a recessed 5'-OH end, which again can serve as a substrate for T7exonuclease, and then the signaling probe is continuously digested from the recessed 5' -OH end, and finally the signaling probe in dsDNA duplex (III) is selectively degraded to release FAM molecules while trigger probe 2 remains intact and is released from dsDNA (III). During autocatalytic cleavage, trigger probe 1 and trigger probe 2 in the dsDNA duplex are protected from T7exonuclease cleavage, since the 5' -OH ends in hairpin probes HP1 and HP2 remain in a protruding state. Thus, the released trigger probe 1 may hybridize to an excess portion of hairpin probe HP2 to induce T7exonuclease to catalyze the first cycle cleavage of hairpin probe HP2 to release a plurality of trigger probes 2, while the released trigger probe 2 may hybridize to a free signaling probe to induce T7exonuclease to catalyze the second cycle cleavage of signaling probe to release a plurality of FAM molecules. Through a two-step cyclic autocatalytic cleavage process, a large number of fluorophore molecules (i.e., FAM) are eventually released from the signaling probe, producing a significantly amplified fluorescent signal. In contrast, hairpin probe HP1 cannot be cleaved by APE1 at the 2' -deoxyinosine site in the absence of hAAG. Hairpin probes HP1 and HP2 with protruding 5' -OH ends can therefore hinder the T7 exonuclease-assisted autocatalytic recycling cleavage process, resulting in no detection of enhanced fluorescent signal. By utilizing the high precision of in vivo DNA repair mechanism, the high specificity of T7exonuclease to catalyze mononucleotide hydrolysis and the high efficiency of autocatalytic recycling amplification reaction, the method disclosed by the invention can be used for simply, conveniently and effectively detecting hAAG activity.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present disclosure, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.
Example 1
DNA repair controls T7 exonuclease-assisted autocatalytic cycle signal amplification: prior to performing the amplification reaction, all synthetic oligonucleotides were dissolved with 1 XTris-EDTA buffer (10 mM Tris), 1 mM EDTA, pH 8.0) to prepare a stock solution, stored at-20℃and then hairpin probes (i.e., HP1 and HP 2) were diluted to 1. Mu. Mol/L with hybridization buffer (1.5 mM magnesium chloride, 10 mM Tris-HCl, pH 8.0) and incubated at 95℃for 5 minutes, followed by slow cooling to room temperature to form hairpin structures. The determination of hAAG activity involves two consecutive reaction steps. First, a volume of 2 microliters of HP1 (1 micromole per liter) was added to the excision reaction system (20 microliters) comprising hAAG,2 microliters of 10 XThermopol reaction buffer, 0.3 units per microliter (U/. Mu.L) of APE1,2 microliters of 10 XNEBuffer 4, and incubated at 37 degrees Celsius for 60 minutes. Next, 5. Mu.l of the above excision product was added to an amplification reaction system (20. Mu.l) comprising 250 nanomoles per liter of HP2, 700 nanomoles per liter of signaling probe, 15 units of T7exonuclease, 2. Mu.l of 10 XNEBuffer 4, and incubated at 25℃for 50 minutes to perform a T7 exonuclease-assisted autocatalytic recirculating amplification reaction in the absence of light.
Fluorescence measurement and gel electrophoresis analysis: 20. Mu.l of the amplification product was first diluted to 60. Mu.l with ultrapure water, and then fluorescence spectrum was scanned in the range of 505 to 620nm by Hitachi F-7000 fluorescence spectrophotometer (Tokyo, japan) at an excitation wavelength of 491nm, and fluorescence intensity at 520nm was recorded for data analysis. For analysis of the excision products and amplification products, 12% non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis was performed in 1 XTBE buffer (9 nanomoles per liter Tris-HCl,9 millimoles per liter boric acid, 0.2 millimoles per liter EDTA, pH 7.9) at a constant voltage at room temperature for 45 minutes using SYBR Gold as a fluorescent indicator and analyzed using a Chemisoc MP imaging system (Hercules, california, U.S.A.).
The sequence of hairpin probe 1 is: 5' -GTA GTG AGG TAG GTT GTA TIG TTG GGT TGA ACT ATA CAA CCT ACC-3' (wherein base I is 2' -deoxyinosine), SEQ ID NO.1
The sequence of hairpin probe 2 is: 5'-TGT ATA GTT CAA CCC GGG ACC TAA GAG CAT TCT ACA CCT CTT AGG TCC CTG C-3', SEQ ID NO.2
The sequence of the signal probe is as follows: 5'-AAG AGG TGT A-3' (wherein the 5 'and 3' ends are modified with a fluorophore FAM and a quencher BHQ1, respectively), SEQ ID NO.3
The sequence of the cleavage fragment is: 5'-GTA GTG AGG TAG GTT GTA T-3', SEQ ID NO.4
The sequence of trigger probe 1 is: 5'-GTT GGG TTG A AC TAT ACA ACC TAC C-3', SEQ ID NO.5
Experimental principle as shown in fig. 1, this example designed two hairpin probes (i.e., HP1 and HP 2) and both had prominent 5'-OH ends, in hairpin probe HP1, a 2' -deoxyinosine (I) damage site was modified at a site 3 bases from the loop on the stem, which could be recognized by hAAG. In the presence of hAAG, it will specifically recognize and cleave hairpin probe HP1 at the 2 '-deoxyinosine (I) site, resulting in hairpin structure expansion, while producing dsDNA duplex (I) with protruding 5' -OH and 5'-dRP ends (one strand of the formed dsDNA duplex (I) with protruding 5' -dRP ends will be partially complementary to a partial region on hairpin probe HP 2). dsDNA duplex (II) with a recessed 5' -OH end is formed by a cohesive end mediated strand displacement reaction (tmmdr). This newly formed dsDNA duplex (II) can serve as a substrate for T7exonuclease, and then hydrolyze specifically from the recessed 5' -OH end in HP2, releasing trigger probe 1 and simultaneously releasing trigger probe 2 in HP 2. Since the signaling probe is complementary to a partial region of trigger probe 2, released trigger probe 2 will hybridize to the signaling probe to form a dsDNA duplex (III) with a recessed 5'-OH end, which again can serve as a substrate for T7exonuclease, and then continue to digest the signaling probe from the recessed 5' -OH end, eventually the signaling probe in dsDNA duplex (III) is selectively degraded to release FAM molecules while trigger probe 2 remains intact and is released from dsDNA (III). Through a two-step cyclic autocatalytic cleavage process, a large amount of fluorescent molecules (i.e., FAM) are finally released from the signaling probe, producing a significantly amplified fluorescent signal. In contrast, in the absence of hAAG, the 2' -deoxyinosine damage site in hairpin probe HP1 could not be recognized, and hairpin probe HP1 was not cleaved by APE 1. Hairpin probes HP1 and HP2 with protruding 5' -OH ends therefore block the T7 exonuclease-assisted autocatalytic recycling cleavage process and fail to generate enhanced fluorescent signals.
Experimental verification of principle
To demonstrate the feasibility of the disclosure, the present example selected non-denaturing gel electrophoresis to analyze the products. As shown in FIG. 2A, in the presence of APE1+HP1, only one band (FIG. 2A, lane 4) was observed at the same position as hairpin probe HP1 alone (45 nt) (FIG. 2A, lane 3), indicating that cleavage of hairpin probe HP1 could not be induced without hAAG. In the presence of haag+ape1+hp1, characteristic bands of 19nt and 25nt were detected (fig. 2A, lane 5), which is the size of 19nt cleavage products (fig. 2A, lane 1) and 25nt trigger probe 1 (fig. 2A, lane 2), demonstrating that hAAG can specifically cleave 2' -deoxyinosine and induce APE 1-mediated cleavage of HP1 to produce two short DNA fragments (i.e., 19nt cleavage product and 25nt trigger probe 1). Furthermore, in order to investigate the T7 exonuclease-assisted autocatalytic recycling cleavage process, 5. Mu.L of the cleavage product described above (FIG. 2A, lanes 4 and 5) was added to a 20. Mu.L amplification reaction system containing HP2, signaling probe and T7exonuclease, and an autocatalytic recycling cleavage reaction was performed. After the reaction was terminated, the reaction products were further analyzed using non-denaturing gel electrophoresis. As shown in FIG. 2B, in the absence of hAAG, only the band of HP2 (52 nt) was detected, which means that no cleavage reaction occurred (FIG. 2B, lane 1). In the presence of hAAG, another characteristic band (25 nt) was observed (FIG. 2B, lane 3), which was completely identical to the size of the synthesized trigger probe 1 (25 nt) (FIG. 2B, lane 2). The experimental results indicate that 25nt trigger probe 1 (i.e., the cleavage products of hAAG and APE1 catalyzed HP 1) can hybridize to the HP2 portion via TMSDR to initiate T7exonuclease catalyzed recirculation cleavage of HP2 to release a large amount of trigger probe 2 (25 nt, FIG. 2B, lane 3). Thus, the gel electrophoresis experiments described above (fig. 2A and 2B) clearly demonstrate that hAAG and APE1 can catalyze cleavage of hairpin probe HP1 at the 2' -deoxyinosine site to produce 25nt trigger probe 1, which can successfully induce subsequent T7 exonuclease-assisted autocatalytic recycling HP2 cleavage, releasing trigger probe 1. More importantly, in the presence of ape1+hp1 (fig. 2A, lane 4), haag+ape1+hp1 (fig. 2A, lane 5) and haag+ape1+t7 exonuclease+hp1+hp2 (fig. 2B, lane 3), no additional bands were shown, demonstrating the high specificity of DNA repair-controlled T7 exonuclease-assisted autocatalytic recirculation amplification, with no non-specific DNA fragment generation. The high specificity can be attributed to these three factors: (1) high accuracy of DNA repair mechanisms in vivo, (2) high specificity of hAAG-catalyzed 2' -deoxyinosine excision, and (3) high resolution of T7 exonuclease-catalyzed single base mismatches.
Sensitivity experiment
Under optimal experimental conditions, the sensitivity of the present disclosure to detect human alkyl adenine DNA glycosylase activity was evaluated, and analytical assays were performed at various concentrations in this example, the results of which are shown in fig. 3. With hAAG concentration from 1X 10 -5 The increase in U/. Mu.L to 0.1U/. Mu.L correspondingly increased the fluorescence intensity, indicating that the increase in fluorescence signal is highly dependent on the concentration of hAAG. The logarithm of the fluorescence intensity and the hAAG concentration shows good linear relation in a certain concentration range, and the linear regression equation is F=5137.9+901.8log 10 C, correlation coefficient 0.9942, wherein F represents fluorescence intensity, C represents hAAG concentration (U/. Mu.L), and detection limit was calculated to be as low as 4.9X10 -6 U/μL。
Specificity experiments
To demonstrate the good selectivity of the present disclosure, the present example compares the fluorescence intensities of non-specific proteins, such as Bovine Serum Albumin (BSA) and immunoglobulin G (IgG), 8-oxyguanine-DNA glycosylase (alogg 1), and hAAG. As shown in fig. 4, only weak fluorescent signals were observed in the presence of BSA, igG and alogg 1 and reaction buffer. Only in the presence of hAAG can a high fluorescent signal be detected. The experimental results show that only hAAG can specifically recognize and cleave the hairpin substrate (i.e., hairpin probe HP 1) at the 2' -deoxyinosine site to induce T7 exonuclease-assisted autocatalytic recycling signal amplification. Thus, the present disclosure can resist interference of other proteins, with good specificity.
The foregoing description of the preferred embodiments of the present disclosure is provided only and not intended to limit the disclosure so that various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
SEQUENCE LISTING
<110> Shandong university of teachers and students
<120> sensor and method for detecting hAAG by controllable autocatalytic cleavage-mediated fluorescence recovery
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Claims (7)
1. A sensor for detecting hAAG based on controllable autocatalytic cleavage-mediated fluorescence recovery, comprising hairpin probe 1, hairpin probe 2, signaling probe, human apurinic/apyrimidinic endonuclease and T7 exonuclease;
hairpin probe 1 is DNA of a stem-loop structure with a protruding 5'-OH end, the stem of the 5' -OH end of hairpin probe 1 modifies a 2 '-deoxyinosine capable of being recognized by hAAG, the 2' -deoxyinosine is positioned at a position 3 bases away from the loop, hairpin probe 1 contains trigger probe 1, trigger probe 1 is a DNA sequence from 2 '-deoxyinosine to the 3' -end, trigger probe 1 contains a first DNA sequence capable of being complementary to a second DNA sequence of hairpin probe 2, and the first DNA sequence is a DNA sequence between the 2 '-deoxyinosine and a base position paired with the 2' -deoxyinosine;
hairpin probe 2 is DNA with a stem-loop structure with a protruding 5'-OH end, hairpin probe 2 is composed of trigger probe 2 and a second DNA sequence, the second DNA sequence is a DNA sequence with a protruding 5' -OH end, trigger probe 2 contains a third DNA sequence which can be complementary with a signal probe, one part of the third DNA sequence is positioned on the stem, and the other part of the third DNA sequence is positioned on the loop;
the signal probe is single-stranded DNA, and the two ends of the signal probe are respectively modified with a fluorophore and a quencher;
the fluorophore and the quencher are respectively modified at the 5 'end and the 3' end of the signaling probe;
the fluorophore is FAM, and the quencher is BHQ1;
the sequence of hairpin probe 1 is: 5'-GTA GTG AGG TAG GTT GTA TIG TTG GGT TGA ACT ATA CAA CCT ACC-3';
the sequence of hairpin probe 2 is: 5'-TGT ATA GTT CAA CCC GGG ACC TAA GAG CAT TCT ACA CCT CTT AGG TCC CTG C-3';
the sequence of the signal probe is as follows: 5'-AAG AGG TGT A-3'.
2. Use of a sensor for detection of hAAG based on controllable autocatalytic cleavage-mediated fluorescence recovery in the preparation of a kit for detection of hAAG, characterized in that the sensor of claim 1 is provided; when hAAG exists, hAAG and APE1 specifically recognize and cleave hairpin probe 1 at the 2' -deoxyinosine site of hairpin probe 1, so that hairpin probe 1 expands hairpin structure, dsDNA duplex I with protruding 5' -OH and 5' -dRP ends is generated, protruding 5' -dRP ends in dsDNA duplex I are hybridized with a second DNA sequence of hairpin probe 2 through a strand displacement reaction mediated by sticky ends, a first DNA sequence of trigger probe 1 is hybridized with a signal probe to form dsDNA duplex II with recessed 5' -OH ends, dsDNA duplex II is used as a substrate of T7exonuclease, recessed 5' -OH ends in hairpin probe 2 are specifically hydrolyzed under the action of T7exonuclease, trigger probe 2 is released while trigger probe 2 in hairpin probe 2 is released, released trigger probe 1 is hybridized with excessive hairpin probe 2, T7exonuclease is induced to catalyze the first cycle cleavage of hairpin probe HP2 to release a plurality of trigger probes 2, a third DNA sequence of trigger probe 2 is hybridized with a signal probe to form dsDNA duplex II with recessed 5' -OH ends, dsDNA duplex III is formed as a substrate of T7exonuclease, and thus signal probe III is released from the second exonuclease, and signal III is released from the probe 2 by the exonuclease, thereby the signal III is continuously cleaved with the probe 2, and the signal III is released.
3. The use according to claim 2, characterized by the steps of:
(1) Hairpin probe 1 was added to a first solution containing hAAG, 10 XThermopol reaction buffer, APE1, 10 XNEBuffer 4 and incubated with heating;
(2) Adding the material obtained in the step (1) into a second solution containing hairpin probes 2, signal probes, T7exonuclease and 10 XNEBuffer 4 for incubation;
(3) And (3) performing fluorescence detection on the incubated material in the step (2).
4. The use according to claim 3, wherein the incubation conditions in step (1) are: the temperature is 37 plus or minus 0.5 ℃ and the time is 60 to 90 minutes;
or, in the first solution of step (1), the concentration of APE1 is 0.3 units per microliter; every 20 microliters of the first solution contains 2 microliters of 10 XThermopol reaction buffer, 2 microliters of 10 XNEBuffer 4;
or, in the step (1), the volume ratio of the solution of the hairpin probe 1 to the first solution is 1:9.5-10.5, and the concentration of the hairpin probe 1 in the solution of the hairpin probe 1 is 1 micromole per liter.
5. The use according to claim 3, wherein the incubation conditions in step (2) are: the temperature is 25+/-0.5 ℃ and the time is 50-60 min;
or, in the second solution of step (2), the hairpin probe 2 has a concentration of 250 nanomoles per liter, the signaling probe has a concentration of 700 nanomoles per liter, and the T7exonuclease has a concentration of 15 units per 20 microliters, each 20 microliters of the second solution containing 2 microliters of 10 x NEBuffer 4;
or in the step (2), the volume ratio of the material in the step (1) to the second solution is 1:4.5-5.5;
or, in the step (3), the detection excitation wavelength is 491nm and the emission wavelength is 520nm.
6. Use according to claim 2, characterized in that single-stranded hairpin probe 1 and/or single-stranded hairpin probe 2 is diluted with hybridization buffer, heated to 95 ℃ and incubated for 5min, and then cooled to room temperature, single-stranded hairpin probe 1 and/or single-stranded hairpin probe 2 forming a stem-loop structure.
7. A kit for detecting hAAG comprising the sensor of claim 1 and a buffer solution.
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