CN111676269A

CN111676269A - Nucleic acid nano-structure probe and preparation method and application thereof

Info

Publication number: CN111676269A
Application number: CN202010544295.4A
Authority: CN
Inventors: 钱永忠; 潘烨灿; 苏昕; 翁瑞; 邱静
Original assignee: Institute of Agricultural Quality Standards and Testing Technology for Agro Products of CAAS
Current assignee: Institute of Agricultural Quality Standards and Testing Technology for Agro Products of CAAS
Priority date: 2020-06-15
Filing date: 2020-06-15
Publication date: 2020-09-18
Anticipated expiration: 2040-06-15
Also published as: CN111676269B

Abstract

The invention discloses a nucleic acid nano-structure probe and a preparation method and application thereof, and relates to the technical field of molecular detection, wherein the nano-nucleic acid probe comprises a nano-molecular cage with a tetrahedral structure and formed by 4 main chain DNA single-strands, wherein 2 main chain DNA single-strands are respectively provided with a branched chain α and a branched chain β which extend out of the tetrahedral structure, b 2 non-main chain DNA single-strands which are combined with the branched chain α through base complementary pairing, one of the non-main chain DNA single-strands is modified with a fluorescent group, the other is modified with an AP (AP) site and a quenching group, and c, the other 1 of the non-main chain DNA single-strands which are combined with the branched chain β through base complementary pairingThe non-backbone DNA single strand of (1). The invention also discloses a preparation method and application of the nucleic acid nano structure. The nucleic acid nano-probe can simultaneously detect APE1 and O in cells₂·^‑The method can be used for simultaneously measuring the nucleic acid repair enzyme and the active oxygen small molecule. By confocal imaging instrument, target detection objects APE1 and O in cells can be detected₂·^‑Detection is performed over a spatial distribution.

Description

Nucleic acid nano-structure probe and preparation method and application thereof

Technical Field

The invention relates to the technical field of molecular detection, in particular to a nucleic acid nano-structure probe and a preparation method and application thereof.

Background

The base excision repair pathway (BER) in cells is a key step in response to oxidative stimuli and has special significance in maintaining the stability of cell genomes. Apurinic/Apyrimidic (AP) endonuclease, APE1, is a multifunctional protein that is a key enzyme involved in the recognition and processing of AP sites in the BER pathway and plays an important role in maintaining the intracellular environmental balance. When the redox system of the cell is unbalanced under endogenous or exogenous stimulation, the APE1 can directly or indirectly regulate the redox state of the cell, thereby helping to maintain the steady state of the cell. APE1 has redox activity and base repair activity, and is also called redox factor (Redox-1, Ref-1). In addition, APE1 is a multifunctional enzyme. The N-terminal domain of APE1 has redox regulatory activity and the C-terminal domain, which determines DNA repair activity, is located near the N-terminal domain. APE1 can redox many transcription factors, thereby participating in a variety of important cellular response processes.

Superoxide anion (O)₂·^-) Is the main precursor of most ROS (reactive oxygen species) and is formed by the reduction of one electron of a triplet dioxygen molecule. In the presence of hydrogen ions (H)⁺) In the presence of O₂·^-Mutation to hydrogen peroxide (H)₂O₂) And further reduced to hydroxyl radical (. OH) or water. H⁺And O₂·^-Are associated with the induction and growth of a variety of diseases, including inflammation, ischemia-reperfusion injury, neurodegenerative diseases, and cancer. O in cells₂·^-Will be too severeThe expression result of APE1 enzyme in cells is seriously influenced, and the redox level and the enzyme expression pathway of the organism are changed under the exogenous stimulation. Thus, study of APE1 and O₂·^-The effect of expression in vivo is particularly important, especially by studying APE1 and O in vivo₂·^-The time distribution difference and the space distribution difference have practical significance for the research of most oxidation damage directions and metabolic toxicology directions in the future.

Although some methods can detect APE1 and ROS in vitro and in vivo, APE1 and O in cells are difficult to detect₂·^-Simultaneous identification, localization and quantification are performed. In recent years, DNA nanostructures have gained increasing attention and are widely used in biological assays due to their ease of synthesis, good rigidity and stability, their resistance to degradation by nucleases, their ability to cross membranes, and their programmability. The monodispersed structure and high biocompatibility of the DNA nano material based on the stereo structure enable the probe to be internalized into mammalian cells through endocytosis, so that intracellular imaging is realized. In particular, the kit has high-efficiency and specific detection capability and can resist the damage of a super-rigid structure. Therefore, the development of a special DNA nanostructure-based method for simultaneously detecting APE1 and O in cells is urgently needed₂·^-The research tool of (1).

Disclosure of Invention

The invention provides a nucleic acid nano-structure probe, and the nucleic acid nano-structure probe constructed by the invention can quantitatively detect and position APE1 and O under different oxidative stresses in a cell experiment₂·^-The method is convenient to be applied to simultaneous determination of the nucleic acid repair enzyme and the active oxygen micromolecules. The invention also provides a preparation method and application of the nucleic acid nano-structure probe and a method for detecting by using the nucleic acid nano-structure probe.

The invention is realized by the following technical scheme:

in one aspect, the present invention provides a nucleic acid nanostructure probe comprising:

a. the nano molecular cage with a tetrahedral structure is formed by 4 main chain DNA single chains; wherein 2 main chain DNA single chains respectively have a branched chain alpha and a branched chain beta extending out of a tetrahedral structure;

b. 2 non-main chain DNA single-strands combined with the branched chain alpha through base complementary pairing, wherein one is modified with a fluorescent group, and the other is modified with an AP locus and a quenching group;

c. and (b) another 1 single strand of non-backbone DNA comprising ethidium dihydrogen bonded via base-complementary pairing to said branched chain β.

In a specific embodiment, in 2 non-main chain DNA single strands combined with the branched chain alpha through base complementary pairing, the 5 'end of one strand is modified with a fluorescent group, the 3' end of the other strand is modified with a quenching group, and an AP locus is arranged at a position 8-10 bp adjacent to the quenching group; the fluorescent group and the quenching group on 2 non-main chain DNA single chains are adjacent in position;

the fluorescent group is selected from JOE, HEX, VIC, ROX, CY3 or CY5, and the quenching group is selected from BHQ1, BHQ2 or BHQ 3;

preferably, the fluorescent group is CY5 and the quencher group is BHQ 3.

In a specific embodiment, the sequences of the backbone DNA single strands having said branched chain α and said branched chain β are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

In the following of the present invention, SEQ ID NO.1 and SEQ ID NO.2 are also referred to as P1 and P2.

In a specific embodiment, the sequences of the other 2 backbone DNA single strands constituting the tetrahedral structure are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

Hereinafter, SEQ ID NO.3 and SEQ ID NO.4 are also referred to as P3 and P4 in the present invention.

In a specific embodiment, the number of bases of the single-stranded DNA having a fluorescent group in the other 2 non-backbone single-stranded DNA bound to the branched chain alpha by base complementary pairing is not less than 10bp, and the number of bases of the single-stranded DNA having a quencher group is not less than 20 bp; preferably, the sequence is shown in SEQ ID NO.5 and SEQ ID NO.6, and X in SEQ ID NO.6 represents a gap, i.e., an abasic site.

In the following of the present invention, the DNA single strand of SEQ ID NO.5 having a CY5 fluorophore is also referred to as P5-CY 5; the DNA single strand of SEQ ID NO.6 with the BHQ3 quencher is also referred to as P6-BHQ 3.

In a specific embodiment, the sequence of the other 1 non-backbone DNA single strands bound to the branched chain β by base complementary pairing is shown in SEQ ID NO.7, and the 5' end of the sequence of SEQ ID NO.7 is linked to ethidium dihydroxide by-NH-CO-.

Specifically, the backbone structure of the nucleic acid nanostructure probe of the present application is synthesized by annealing 4 DNA single strands (named P1, P2, P3, and P4, respectively) with different base lengths through gradient temperature.

In a specific embodiment, 2 main chain DNA single chains respectively have a branched chain alpha and a branched chain beta extending out of a tetrahedral structure, and the base length of the main chain DNA single chain with the branched chain alpha is 103 bp; the length of each of the 2 non-backbone DNA single strands to which the branched chain alpha is bound by base complementary pairing may be 20 bp.

In a specific embodiment, the base length of the backbone DNA single strand having the branch β is 83bp, and the other 1 non-backbone DNA single strand bound to the branch β by base complementary pairing is bound to a chemiluminescent substance capable of detecting a superoxide anion, and preferably, the other 1 non-backbone DNA single strand bound to the branch β by base complementary pairing is bound to a chemiluminescent substance dihydroethidium (HE) at 5' end by an amide bond.

In a specific embodiment, the other 1 single strand of non-backbone DNA comprising ethidium dihydroxide to which said branch β is bound by base complementary pairing has a base length of 20 bp.

In another aspect, the present invention provides a method for preparing the nucleic acid nanostructure probe, comprising the steps of:

(a) designing and synthesizing 4 main chain DNA single chains forming a DNA tetrahedral structure, wherein the length of 2 main chain DNA single chains is longer than that of the other 2 main chain DNA single chains, and the extended parts are a branched chain alpha and a branched chain beta respectively; synthesizing 3 non-backbone DNA single strands according to the sequences of the branched chain alpha and the branched chain beta; wherein 2 are complementary to branched chain alpha bases and 1 is complementary to branched chain beta bases; one of the chains complementary to the branched chain alpha base is modified with a fluorescent group, and the other chain is modified with an AP locus and a quenching group; a carboxyl group is modified at the 5' end of the strand which is complementary with the branched chain beta base;

(b) activating carboxyl on a chain modified with a carboxyl group at the 5' end, adding ethidium dihydrogen, stirring, and then carrying out solid-liquid separation; adding buffer solution to obtain a single chain with the 5' end connected with ethidium dihydrogen through-NH-CO-;

(c) mixing the DNA single chain with the 5' end connected with ethidium dihydrogen through-NH-CO-and other DNA single chains in a buffer solution at the same final molar concentration, heating and denaturing at 80-85 ℃ for 8-10min, and then keeping at 4 ℃ for more than 30min to obtain a DNA tetrahedral structure;

preferably, the denaturation is carried out by heating at 80 ℃ for 10 min.

In one embodiment, in the method for preparing the nucleic acid nanostructure probe, the fluorescent group in the step (a) is selected from JOE, HEX, VIC, ROX, CY3 or CY5, and the quencher group is selected from BHQ1, BHQ2 or BHQ 3; preferably, the fluorescent group is CY5, and the quencher group is BHQ 3;

in the step (b), the step of activating the carboxyl on the chain modified with one carboxyl group at the 5 'end is to mix and stir the chain modified with one carboxyl group at the 5' end and EDC in a buffer solution for 30-40min under the condition of keeping out of the light to activate the carboxyl;

the molar concentration ratio of the DNA single chain modified with a carboxyl group at the 5' end, EDC and ethidium dihydrogen is 1: 10-100: 1-10, preferably 1: 30: 4; preferably, the ethidium dihydrogen is added and stirred for being protected from light, and the stirring is more preferably carried out for 12 to 14 hours;

preferably, the solid-liquid separation is solid-liquid separation by a centrifugal mode, and more preferably, centrifugation is carried out by an ultrafiltration tube; preferably, the centrifugation conditions are above 8000rpm, 4-8min, more preferably 12000rpm, 5 min;

preferably, the buffer is PBS buffer, more preferably 1 x PBS buffer.

In one embodiment, the method for preparing the nucleic acid nanostructure probe comprises the following steps:

(1) designing and synthesizing 4 main chain DNA single chains with sequences respectively shown as SEQ ID NO.1-4 and 3 non-main chain DNA single chains with sequences respectively shown as SEQ ID NO. 5-7; wherein the 5' end of SEQ ID NO.7 is modified with a carboxyl group; SEQ ID NO.5-6 are respectively modified with a fluorescent group and a quenching group;

(2) mixing the DNA single chain of SEQ ID NO.7 modified with a carboxyl group at the 5' end with EDC in a buffer solution under the dark condition, stirring for 30-40min to activate the carboxyl group, adding ethidium dihydrogen, stirring for 12-14h in the dark condition, and centrifuging by using an ultrafiltration tube; adding buffer solution to obtain a single chain of SEQ ID NO.7 with the 5' end connected with ethidium dihydrogen through-NH-CO-;

preferably, the ultrafiltration tube is a 0.5mL 3K ultrafiltration centrifuge tube;

preferably, the centrifugation conditions are above 8000rpm, 4-8min, more preferably 12000rpm, 5 min;

preferably, the buffer is PBS buffer, more preferably 1 x PBS buffer;

(3) mixing the DNA single chains shown in SEQ ID NO.1-6 and the DNA single chain of SEQ ID NO.7 of which the 5' end is connected with ethidium dihydrogen through-NH-CO-in a buffer solution at the same final molar concentration, heating and denaturing at 80-85 ℃ for 8-10min, quickly reducing to 4 ℃ and keeping for more than 30min to obtain a DNA tetrahedral structure;

preferably, the denaturation is carried out by heating at 80 ℃ for 10 min.

In the following of the present invention, the DNA single strand of SEQ ID NO.7 modified at the 5' end with a carboxyl group is also referred to as P7-COOH; and the DNA single strand of SEQ ID NO.7 linked at the 5' end to ethidium dihydroxide via-NH-CO-is also referred to as P7-COOH-HE or P7-HE.

In the context of the present invention, EDC means 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, a water-soluble carbodiimide, which is used as a carboxyl activating reagent in amide synthesis.

In a specific embodiment, the fluorescent group is selected from JOE, HEX, VIC, ROX, CY3, or CY5, and the quenching group is selected from BHQ1, BHQ2, or BHQ 3; preferably, the fluorescent group is CY5, and the quencher group is BHQ 3;

in the step (2), the molar concentration ratio of the DNA single strand of SEQ ID NO.7 modified at the 5' end with a carboxyl group, EDC and HE is 1: 10-100: 1-10, preferably 1: 30: 4.

preferably, in the aforementioned step (2), the concentration of the DNA single strand of SEQ ID NO.7 modified at the 5' -end with a carboxyl group is 100nM to 100. mu.M, preferably 25. mu.M.

In the preparation method of the nucleic acid nano-structure probe, a DNA single chain with a 5' end connected with ethidium dihydrogen through-NH-CO-and other DNA single chains need to be mixed in a buffer solution at the same final molar concentration, the DNA single chains are mixed under the condition that the final molar concentration of each DNA single chain is the same, each side of a tetrahedron is composed of two sections of DNA single chains, the two sections of DNA chains on each side of the tetrahedron are complementarily combined, and each side of the tetrahedron formed by the single chains is constructed into a three-dimensional solid through base complementary pairing.

In a proper amount of Mg²⁺When present, the single DNA strands can self-assemble to form stable DNA nanostructures through gradient temperature annealing. In the specific embodiment of the invention, a DNA single chain containing a fluorescent group, a quenching group and an AP site and carrying a fluorescent substance HE and 4 DNA single chains with different base lengths which form the main chain structure of the nucleic acid nano-structure probe are subjected to gradient temperature annealing and assembled into a nano-probe with a tetrahedral stereo structure by a base complementary pairing mode.

The key of the part of the probe which plays a role is a branched chain alpha, 2 non-main chain DNA single strands which are combined with the branched chain alpha through base complementary pairing, and the other 1 non-main chain DNA single strand which comprises ethidium dihydrogen and is combined with the branched chain beta through base complementary pairing. The result of probe synthesis can be characterized by polyacrylamide gel electrophoresis.

When meeting and binding with a target substance APE1, the AP site is broken, so that a single-stranded nucleic acid containing a quenching group (such as BHQ3) is released, and a fluorescent group CY5 is exposed and shows fluorescence; when meeting soldiers combined with active oxygen molecule superoxide anion O₂·^-In this case, the dihydroethidium on the non-backbone DNA single strand containing dihydroethidium is dehydrogenated to form an oxidized form of ethidium, which fluoresces.

In a specific embodiment, the GC content in the non-backbone DNA single strand modified with a fluorophore at the 5' end is preferably in the range of 30% to 50%, and too high GC content will result in reduced detection accuracy.

In one embodiment of the present invention, the nucleic acid nanostructure probe is prepared as follows:

(1) ethidium dihydroxide (HE) is bonded to P7-COOH via an amide bond. The single-stranded P7-COOH (25 μ M) was removed and mixed with EDC (750 μ M) in a buffered solution of 1 × PBS (PH 7.4) under exclusion of light and stirred for 30min to activate the carboxyl group. Subsequently, HE (100. mu.M) was added to the above solution and stirred for 12h in the absence of light. The above preparation was centrifuged at 12000rpm for 5min using an ultrafiltration tube (3k, 0.5 ml). Adding a proper amount of PBS buffer solution to obtain a target substance p7-COOH-HE (25 mu M), and standing at-20 ℃ for later use.

(2) The remaining DNA single strands P1, P2, P3, P4, P5-CY5, and P6-BHQ3 were diluted to 25. mu.M with enzyme-free water and stored in a refrigerator at-20 ℃ until use.

(3) The 7 DNA single strands diluted in advance were taken out from a refrigerator at-20 ℃ and vortexed for 10 seconds to mix the single strands thoroughly.

(4) To an EP tube was added 31. mu.L of enzyme-free water, 5. mu.L of 10 × TAE buffer-Mg²⁺2 μ L P1, 2 μ L P2, 2 μ L P3, 2 μ L P4, 2 μ L P5-CY5, 2 μ L P6-BHQ3, 2 μ L P7-COOH-HE; adding the sample on the side wall of the centrifuge tube, adding the sample, and centrifuging and mixing uniformly.

A gradient temperature annealing program (80 ℃ for 10min, 4 ℃ for 30min) is set on a PCR instrument, and the product is stored at 4 ℃ for later use.

The concentration of the synthesized nucleic acid nanoprobe was 50. mu.L.times.1. mu.M, and it was stored in a refrigerator at 4 ℃ in the dark.

In another aspect, the present invention provides a detection method using the nucleic acid nanostructure probe, the detection method including:

when inoculating living cells, reacting 8000-12000 cells with nucleic acid nanostructure probe with concentration of 80-120nM/L, preferably 100 nM/L;

the action time is at least 30 min.

In one embodiment of the present invention, CY5 has an excitation wavelength of 625nm and an emission wavelength of 668 nm; the HE has an excitation wavelength of 488nm and an emission wavelength of 580 nm.

In some embodiments, other cellular dyes may also be used to localize cells in the detection method, such as nuclear or membrane dyes, e.g., Hoechst, and the like.

The application of the nucleic acid nano-structure probe in biological detection, in particular to the application in simultaneously detecting APE1 and active oxygen molecules in cells.

In some embodiments, the cell is a living cell selected from a cell highly expressing the APE1 enzyme, such as a Hela cell, an a549 cell, and the like.

In other embodiments, the method for detecting a nucleic acid nanostructure probe of the present invention further comprises: and (3) recovering, culturing and cryopreserving the cells to be detected. The cell recovery, culture and cryopreservation are carried out according to a conventional method or according to the instruction of the purchased cells.

According to the invention, the result is obtained by applying the probe to the cells for about 30 min. If the cells to be tested contain the target substance APE1 enzyme and superoxide anion O₂·^-In this case, the probe can measure its position and can also quantitatively detect the fluorescence intensity. Wherein, the fluorescent marker CY5 for tracking APE1 enzyme has an excitation wavelength of 625nm and an emission wavelength of 668 nm; for tracing superoxide anion O of active oxygen molecule₂·^-The excitation wavelength of the DNA-ethidium dihydrogen phosphate is 488nm, and the emission wavelength of the DNA-ethidium dihydrogen phosphate is 580 nm.

The nucleic acid nano-structure probe constructed by the invention is a DNA nano-fluorescence detection probe, is based on a tetrahedral nano model with a rigid structure, and consists of 7 DNA single chains with complementary basic groups; the form is a main framework DNA tetrahedral nano material containing two branched chains simultaneously. The AP locus contained in the nucleic acid nano-structure probe can definitely identify APE1 and is cut and broken by APE1 enzyme, and the detection result can be displayed through a fluorescent group. The AP locus and the quenching group BHQ3 are in the same DNA single strand, and the distance is 8-10 bp. In the nucleic acid nano-structure probe, the distance between the AP locus and the quenching group is 8-10 bp, so that the detection accuracy of the nucleic acid nano-structure probe is ensuredWhen the spacing is too small, there is a possibility that the AP site will break prematurely before APE1 recognizes; when the spacing is too large, it is possible that the AP site will not be cleaved after recognition by APE 1. The fluorescent group and the quenching group on the two single nucleic acid strands are not separated (i.e., adjacent), and the probe does not generate a fluorescent signal. When APE1 is encountered, the AP site of the recognition site is broken, a single-stranded nucleic acid containing a quenching group BHQ3 is released, and a fluorescent group CY5 is exposed, so that the probe shows fluorescence. Dihydroxyethylidene (HE) contained in the nucleic acid nanostructure probe is a chemical fluorescent substance for identifying active oxygen, is connected to a DNA single strand through an amide bond, and meets superoxide anion (O) in cells₂·^-) After the specific reaction, the fluorescence at 580nm increased. The DNA single chain (P7-COOH-HE) of SEQ ID NO.7 modified with a carboxyl group at the 5' end activates the carboxyl group through EDC, and is linked with ethidium dihydroxide HE through amido bond, so that active oxygen molecules in cells can be effectively detected, and the active oxygen molecules can react with superoxide anion O₂·^-Has specific recognition capability, which is mainly caused by the fact that ethidium bromide generated after the dehydrogenation of ethidium dihydroxide HE has fluorescence.

The nucleic acid nano-structure probe of the invention is specially designed for the DNA composition sequence of tetrahedrons, can control the structure and the size of DNA tetrahedrons, has good stability and can stably exist in a cell environment. The nucleic acid nano-structure probe can be actively taken up by cells, has high cell entering efficiency and stably exists in the cells for more than 48 hours.

The nucleic acid nano probe constructed by the invention can realize the specific detection of a target detection object and has the characteristics of sensitivity, high efficiency and low detection limit. And the target detection object can be positioned and quantitatively analyzed according to the fluorescence intensity.

The preparation method of the nucleic acid nanoprobe provided by the invention has the advantages of short synthesis time, simple method and easy operation. In the preparation method of the nucleic acid nano probe, firstly, ethidium dihydroxide HE is combined with a DNA single chain (namely P7-COOH) of SEQ ID NO.7, the 5 'end of which is modified with a carboxyl group, through an amido bond to form a DNA single chain (P7-COOH-HE) of SEQ ID NO.7, the 5' end of which is connected with ethidium dihydroxide through-NH-CO-, and after filtration and centrifugation are carried out by using an ultrafiltration tube, P7-COOH-HE is self-assembled with the rest single chains. In addition, the denaturation temperature for probe assembly of the present application was set at 80 ℃ at which detection accuracy for the synthesized nucleic acid nanoprobes is optimal, since conventional temperatures above 90 ℃ would result in the AP sites in the inventive nucleic acid nanoprobe structure being affected, potentially leading to premature fragmentation prior to APE1 recognition.

APE1 and O₂·^-The method has an inseparable relationship, and particularly has important significance for detecting the mitochondrial damage in the aspect of research on mitochondrial damage. The nucleic acid nano-structure probe has rigid DNA tetrahedron, and can simultaneously detect APE1 and O in cells₂·^-The DNA tetrahedral nano fluorescent probe realizes APE1 and O under different oxidative stresses at living cell level₂·^-The APE1 and O can be obtained by the localization and quantitative dynamic detection of₂·^-The correlation between them. Has good application prospect. Taking Hela cells as an example, the detection limit of APE1 enzyme can reach 0.10U, and the detection limit of superoxide anion can reach 0.33 mol/L; the detection specificity is good, the detection time is rapid, and the detection can be realized within 30 min.

The method for detecting by using the nucleic acid nano-structure probe has high efficiency, specificity and repeatability, and provides a new methodology basis and a new idea for deeply developing related researches such as toxicological evaluation, nutritional function evaluation and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram of the structure of a nucleic acid nanoprobe (DNA tetrahedral structure with branched chains) of the present invention;

FIG. 2 is a graph showing the results of characterization of probes synthesized in examples of the present invention using polyacrylamide gel electrophoresis, wherein the bands from left to right are lane1: Marker; lane 2: P1; lane 3: P1+ P2; lane 4: P1+ P2+ P3; lane 5: P1+ P2+ P3+ P4; lane 6: P1+ P2+ P3+ P4+ P5; lane 7: P1+ P2+ P3+ P4+ P5+ P6; lane 8, P1+ P2+ P3+ P4+ P5+ P6+ P7; wherein P5, P6 and P7 refer to the result graphs of the respective amplifications of the sequences SEQ ID NO. 5-7;

FIG. 3 is a graph showing the results of in vitro recognition of APE1 enzyme by the probe of the present invention, wherein (A) is a fluorescence intensity time curve (fluorescence intensity curve with time after the probe is contacted with APE1 at various concentrations) when the probe is labeled with FAM (B) is a fluorescence concentration curve (recognition effect of the probe on APE1 at various concentrations) when the probe is labeled with CY5, and the inner graph is the corresponding standard curve;

FIG. 4 shows the in vitro recognition of superoxide anion O by the probe of the present invention₂·^-The results of (A) are graphs in which (A) is the fluorescence effect of HE-ligated single-stranded DNA on different concentrations of XO/XA (xanthine binds to xanthine oxidase and produces superoxide anion), and (B) is a standard curve of response;

FIG. 5 is a diagram showing the observation results of confocal fluorescence microscopy in examples 1 to 5 of the present invention. (A) Hela cells that were not oxidatively stimulated; (B) hela cells stimulated by oxidation for 30 min; (C) GSH (10. mu.g/L) treatment for 30 min; (D) treating with allicin (50 μ M/L) for 30 min; (E)30min GSH +30min PMA treatment; (F) treating with allicin for 30min and PMA for 30 min; (G) treating with PMA for 30min and GSH for 30 min; (H) graph of results of 30min PMA +30min allicin treatment.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. 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. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention. Unless otherwise indicated, the methods referred to in the examples of the present invention are, unless otherwise indicated, conventional techniques and methods of the relevant art. Materials, reagents, equipment and the like used in examples are commercially available unless otherwise specified.

Example 1

In vitro identification of APE1 enzyme using nucleic acid nanoprobes

(I) Synthesis of Probe

(1) Diluting the P1, P2, P3, P4, P5-CY5, P6-BHQ3 and P7-HE seven DNA single strands to 25 mu M with enzyme-free water, and vortexing for 10s to fully mix the single strands.

(2) A50. mu.L system was established, 31. mu.L of enzyme-free water and 5. mu.L of 10 × TAE buffer-Mg were added²⁺2 μ L P1, 2 μ LP2, 2 μ L P3, 2 μ L P4, 2 μ L P5-CY5, 2 μ L P6-BHQ3, 2 μ L P7-HE; adding the sample on the side wall of the centrifuge tube, adding the sample, and centrifuging and mixing uniformly.

(3) The PCR annealing procedure was applied to allow the 7 DNA single strands to associate into a three-dimensional nucleic acid nanostructure. The PCR was programmed as follows: 10min at 80 ℃; 4 ℃ for 30 min. Stored at 4 ℃ for later use.

A schematic of a nucleic acid nanostructure is shown in figure 1; the results of characterizing the probes synthesized in the examples of the present invention using polyacrylamide gel electrophoresis are shown in FIG. 2.

(II) in vitro identification and results

The synthesized probes were reacted with different concentrations of APE1 enzyme in PBS buffer, and the fluorescence response intensities are shown in FIG. 3. The experiment was performed in a Q-PCR instrument. FIG. 3 shows that the initial rate of fluorescence-time curves shows a good linear relationship between probe and APE1 concentration in the range of 0.10-10.0U. The detection limit was determined to be 0.10U.

Example 2

In vitro identification of superoxide anion O using nucleic acid nanoprobes₂·^-

(I) Synthesis of Probe

The method is the same as that of example 1 (A)

(II) in vitro identification and results

The synthesized probes were reacted in PBS buffer with varying concentrations of xanthine/xanthine oxidase combinations, the fluorescence response intensities of which are shown in fig. 4. The experiment was performed in a fluorometer. 1mol of xanthine and 1U of xanthine oxidase can form 0.33mol of superoxide anion. The initial rate of the fluorescence-concentration curve shows a good linear relationship between the probe and the superoxide anion concentration in the range of 0.33-133.33 mol/L. The detection limit was determined to be 0.33 mol/L.

Example 3

Investigation of cellular APE1 expression and Redox levels exposed to PMA Using nucleic acid nanoprobes

PMA (phorbol ester) is a stimulator of reactive oxygen species, and exposure of cells to PMA results in increased levels of reactive oxygen species and an imbalance in redox levels.

(I) Synthesis of Probe

The method is the same as that of example 1 (A)

(II) cell fluorescence experiment

(1) Cell plating: the cells to be detected were taken out from the incubator, washed with 1 XPBS and repeatedly aspirated and blown into the bottom of the flask with 1ml of fresh complete medium using a pipette gun, and the cells were mixed. Aspirate 1ml of cell mixture into a 1.5ml EP tube and centrifuge at 1000rmp for 3 min. Centrifuging, removing supernatant, uniformly blowing and stirring cells by using a fresh complete culture medium, sucking 200 mu L of uniform mixed liquor, pumping the uniform mixed liquor into a groove of a confocal culture dish, diluting cell suspension to 8000-12000 cells/200 mu L, carrying out 5% CO2, and carrying out overnight culture in a constant-temperature incubator at 37 ℃. Hela cells were used in this example.

(2) Exposure experiment: PMA was diluted to 1. mu.L/mL with PBS containing DMSO (note that DMSO content in the dilution should be less than 1%). Add 180. mu.L of fresh phenol red-free medium and 20. mu.L of diluted PMA solution to 1.5mL of EP tube and mix well for further use.

Taking out the confocal culture dish from the constant temperature incubator, sucking the original culture solution, slightly cleaning the surface by using 1 × PBS, discarding the waste liquid, and then injecting the culture solution containing PMA prepared in the step (II) and (2) into the groove of the confocal culture dish in 5% CO₂And culturing in a constant-temperature incubator at 37 ℃ for 0.5 h. The blank was phenol red free medium without PMA.

(3) Sample adding detection, wherein the probe use concentration is 200 muL × 100nM, 180 muL fresh phenol-free medium is sucked into a 1.5mL EP tube, 20 muL 50 muL × 1 muM DNA tetrahedral probe and 1 muL Hoechst 33342 dye (2.5 mug/mL) are added and sucked and uniformly mixed, a confocal culture dish is taken out, the old culture medium is discarded, the mixed culture solution containing the probe is added after 2-3 times of washing by 1 × PBS, and the mixed culture solution is placed in 5% CO₂And performing on-line shooting after incubation in a constant-temperature incubator at 37 ℃ for 30min, wherein the lens is 100 × oil-coated lens, the excitation wavelength of CY5 is 625nm, the emission wavelength is 668nm, the excitation wavelength of P7-COOH-HE is 488nm, the emission wavelength is 580nm, and the emission wavelength of Hoechst 33342 is 350nm and 460 nm.

(4) And (4) observing results: the results are shown in FIG. 5, where (A) untreated Hela; (B) hela (i.e., Hela under the exposure experiment in this example) was stimulated by PMA oxidation for 30 min. According to the detection result of the probe, the graphs (A) and (B) show that the fluorescence intensity of APE1 enzyme and the fluorescence intensity of ROS in Hela after PMA oxidation stimulation are obviously enhanced compared with that of untreated Hela; and the obvious enhancement of the fluorescence intensity of the two has positive correlation in spatial distribution, which shows that the ROS concentration of the PMA treated Hela cells is increased after stimulation, and the redox level is unbalanced, so that more APE1 enzyme is needed for redox repair.

Example 4

Study of cellular APE1 expression and redox levels exposed to GSH using nucleic acid nanoprobes

GSH, also known as glutathione, is involved in a variety of redox reactions in the body, and in this example GSH is a reducing agent for active oxygen.

(I) Synthesis of Probe

The procedure is as in example 1 (one).

(II) cell fluorescence experiment

(1) Cell plating: the procedure was as in (II) (1) of example 3.

(2) The exposure experiment comprises diluting GSH with PBS solution to 100g/L, adding 180 μ L of fresh phenol-free red culture medium and 20 μ L of diluted GSH solution into 1.5mL EP tube, mixing, taking out the confocal culture dish from the incubator, sucking the original culture solution, slightly cleaning the surface with 1 × PBS, discarding the waste liquid, injecting the prepared culture solution containing GSH into the groove of the confocal culture dish (the concentration of GSH in the culture solution is 10g/L), and treating with 5% CO₂And culturing in a constant-temperature incubator at 37 ℃ for 0.5 h. The blank was phenol red-free medium without GSH.

(3) Sample adding detection: the procedure was as in (two) (3) of example 3.

(4) And (4) observing results: the results are shown in FIG. 5, where (A) untreated Hela; (C) hela (i.e., Hela under the exposure experiment in this example) treated with 30min GSH (10. mu.g/L). According to the probe detection result graphs (A) and (C), the fluorescence intensity of APE1 enzyme and the fluorescence intensity of ROS in the Hela treated by 30minGSH (10 mug/L) are obviously weakened compared with that of the untreated Hela, and the change of the fluorescence intensities of the two are in positive correlation on the spatial distribution.

Example 5

Application of nucleic acid nanoprobe to research on influence of Allicin on levels of APE1 enzyme and active oxygen in cells

Allicin, also known as Allicin (C)₆H₁₀S₃Trithio-propylene), a sulfur-containing compound extracted from plants of the genus allium, diallyl thiosulfinate. Has special flavor and certain oxidation resistance.

(I) Synthesis of Probe

The procedure is as in example 1 (one).

(II) cell fluorescence experiment

(1) Cell plating: the procedure was as in (II) (1) of example 3.

(2) Exposure experiment: allicin was diluted to 500. mu.M/L. To a 1.5mL EP tube, 180. mu.L of freshTaking out the confocal culture dish from the constant-temperature incubator, sucking original culture solution, slightly cleaning the surface by using 1 × PBS, discarding waste liquid, pumping the prepared culture solution containing the Allicin into a groove of the confocal culture dish (the concentration of the Allicin in the culture solution is 50 mu M/L) in 5% CO₂And culturing in a constant-temperature incubator at 37 ℃ for 0.5 h. The blank control was phenol red-free medium without Allicin.

(3) Sample adding detection: the procedure was as in (two) (3) of example 3.

(4) And (4) observing results: the results are shown in FIG. 5, where (A) untreated Hela; (D) hela (i.e., Hela under the exposure experiment in this example) treated with allicin (50. mu.M/L) for 30 min. According to the probe detection results, the graphs (A) and (D) show that the fluorescence intensity of APE1 enzyme and the fluorescence intensity of ROS in Hela after 30min of allicin (50 mu M/L) are obviously weakened compared with that of untreated Hela, and the change of the fluorescence intensities of the two have positive correlation in spatial distribution.

Example 6

Application of nucleic acid stereo nano probe to research on therapeutic effect of Allicin on cellular redox and enzymatic activity level

(I) Synthesis of Probe

The procedure is as in example 1 (one).

(II) cell fluorescence experiment

(1) Cell plating: the procedure was as in (II) (1) of example 3.

(2) PMA stimulant exposure experiments: the procedure was the same as in example 3 (two) (2), and at least two groups of culture dishes were prepared.

(3) Repair exposure experiments: a set of dishes were exposed to Allicin as in example 3 (two) (2). The control group was exposed to GSH in the same manner as in example 3 (two) (2).

(4) Sample adding detection: the procedure was as in (two) (3) of example 3.

(5) And (4) observing results: the results are shown in FIG. 5, in which (G) is Hela treated with 30min PMA +30min GSH (i.e., control group of this example); (H) hela treated with 30min PMA +30min allicin (i.e., the repair exposure experimental group of this example). According to the probe detection result graphs (G) and (H), the fluorescence intensity of APE1 enzyme and the fluorescence intensity of ROS in Hela treated with 30min PMA +30min allicin (i.e., the repair exposure experimental group of this example) are obviously weakened compared with the control group, and the change of the fluorescence intensities of the two are in positive correlation on the spatial distribution.

Example 7

Application of nucleic acid nanoprobe to research on protective effect of Allicin on redox and enzymatic activity level of cells

(I) Synthesis of Probe

The procedure is as in example 1 (one).

(II) cell fluorescence experiment

(1) Cell plating: the procedure was as in (II) (1) of example 3.

(2) Antioxidant exposure experiment: a set of dishes were exposed to Allicin as in example 3 (two) (2). The control group was exposed to GSH in the same manner as in example 3 (two) (2).

(3) PMA stimulant exposure experiments: the procedure was as in (two) (2) of example 3.

(4) Sample adding detection: the procedure was as in (two) (3) of example 3.

(5) And (4) observing results: the results are shown in FIG. 5, in which (E) is Hela treated with GSH at 30min + PMA at 30min (i.e., the control group of this example); (F) hela treated with allicin for 30min + PMA for 30min (i.e., the repair exposure experimental group of this example). Cells incubated by glutathione (E) or allicin (F) are stimulated by PMA, the fluorescence intensity is obviously weakened compared with that of the cells (C), and the fluorescence intensity is almost consistent with that of the cells (A) which are not stimulated by oxidation. This indicates that both allicin and glutathione have oxidative protection, which protects cells from oxidative damage. Cells stimulated by PMA are incubated by glutathione (G) or allicin (H), the fluorescence intensity is obviously weakened compared with that of cells (C), and the fluorescence intensity is almost consistent with that of cells (A) which are not stimulated by oxidation. This shows that both garlicin and glutathione have the effect of oxidative repair, so that the cells damaged by oxidation can be recovered to be normal in a short time.

The nucleic acid sequences employed in the examples of the present invention are shown in Table 1.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> institute of agricultural quality standards and testing technology of Chinese academy of agricultural sciences

<120> nucleic acid nano-structure probe and preparation method and application thereof

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<170>PatentIn version 3.3

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Claims

1. A nucleic acid nanostructure probe, characterized in that it comprises:

2. The nucleic acid nanostructure probe according to claim 1, wherein the 5 'end of one of 2 non-backbone DNA single strands bound to the branched chain α through base complementary pairing is modified with a fluorescent group, the 3' end of the other is modified with a quencher group, and an AP site is provided at a position 8-10 bp adjacent to the quencher group; the fluorescent group and the quenching group on 2 non-main chain DNA single chains are adjacent in position;

preferably, the fluorescent group is CY5 and the quencher group is BHQ 3.

3. The nucleic acid nanostructure probe of claim 1, wherein the sequences of the backbone DNA single strands having the branched chain α and the branched chain β are shown in SEQ ID No.1 and SEQ ID No.2, respectively.

4. The nucleic acid nanostructure probe of claim 1, wherein the sequences of the other 2 backbone DNA single strands constituting the tetrahedral structure are shown in SEQ ID No.3 and SEQ ID No.4, respectively.

5. The nucleic acid nanostructure probe according to claim 2, wherein the number of bases of a single-stranded DNA having a fluorescent group among the other 2 non-backbone single-stranded DNAs bound to the branched chain α by base complementary pairing is not less than 10bp, and the number of bases of a single-stranded DNA having a quencher group is not less than 20 bp; preferably, the sequences are shown as SEQ ID NO.5 and SEQ ID NO.6, respectively.

6. The nucleic acid nanostructure probe of claim 1, wherein the sequence of the other 1 non-backbone DNA single strands bound to the branched chain β by base complementary pairing is shown in SEQ ID No.7, and the 5' end of the sequence of SEQ ID No.7 is linked to ethidium dihydrovia-NH-CO-.

7. A method for preparing the nucleic acid nanostructure probe of any one of claims 1 to 6, comprising the steps of:

preferably, the denaturation is carried out by heating at 80 ℃ for 10 min.

8. The method for preparing a nucleic acid nanostructure probe according to claim 7,

the fluorescent group in the step (a) is selected from JOE, HEX, VIC, ROX, CY3 or CY5, and the quenching group is selected from BHQ1, BHQ2 or BHQ 3; preferably, the fluorescent group is CY5, and the quencher group is BHQ 3;

preferably, the buffer is PBS buffer.

9. A detection method using the nucleic acid nanostructure probe of any one of claims 1-6, characterized in that:

when inoculating living cells, reacting 8000-12000 cells with a nucleic acid nanostructure probe with the concentration of 80-120nM, preferably 100 nM;

the action time is at least 30 min.

10. Use of the nucleic acid nanostructure probe according to any one of claims 1 to 6 in biological assays, in particular for simultaneous detection of APE1 and reactive oxygen species in a cell.