A method for detecting 8-OG DNA glycosylase activity mediated by an intramolecular catalytic hairpin self-assembly nanosystem

Abstract

The present invention discloses a method for detecting 8-OG DNA glycosylase activity mediated by an intramolecular catalytic hairpin self-assembly nanosystem, which belongs to the field of biological detection and molecular biology technology. Under the action of 8-OG DNA glycosylase, 8-OG in double-stranded DNA is removed, resulting in instability of double-stranded DNA R-T and release of trigger chain T. Then, the released T can start the intraCHA reaction cycle, so that Cy5 and BHQ2 are separated, Cy5 fluorescence is restored, and efficient FRET occurs. The RI-interCHA nanosystem can amplify and image the activity of 8-OG DNA glycosylase outside cells or inside living cells, with a detection limit of 0.2443U / mL. It cannot be degraded by nucleases in cells to produce false positive signals, and has the advantages of satisfactory biosafety, self-delivery, high biostability and specificity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection and molecular biology, and particularly relates to a method for detecting 8-OG DNA glycosylase activity mediated by an intramolecular catalytic hairpin self-assembly nanosystem. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] CHAs are achieved by catalyzing the hybridization of DNA hairpin reactants. In most CHA systems, the DNA hairpin reactants are free, referred to as free CHA. Alternatively, researchers have recently designed intramolecular catalytic hairpin self-assembly (intraCHA) by immobilizing the DNA hairpin reactants on framework nucleic acids (FNAs). Due to the spatial confinement and three-dimensional structure of FNAs, intraCHA exhibits accelerated reaction rates, enhanced endocytosis, and biostability compared to free CHA in live-cell imaging. IntraCHA has recently been developed for intracellular imaging. However, initiators for intraCHA are primarily limited to nucleic acids, and non-nucleic acid-initiated intraCHA are rarely reported. Only Li et al. designed an intraCHA using the endogenous transmembrane glycoprotein mucin 1 (MUC1) and microRNA-21 as initiators. To link MUC1 and intraCHA, an aptamer targeting MUC1 was introduced. However, the affinity between the aptamer and its target was insufficiently strong, limiting the efficiency of intraCHA initiation.

[0004] DNA repair enzymes are an important class of enzymes responsible for recognizing and correcting DNA damage to maintain genomic integrity, and they possess excellent substrate specificity and catalytic activity. To explore the functions of DNA repair enzymes in complex cellular environments and their clinical relevance, imaging studies of DNA repair enzyme activity in living cells have been conducted based on DNA probes. Notably, amplified imaging of DNA repair enzyme activity in living cells has recently been achieved through the introduction of signal amplification strategies. However, these amplified imaging methods require the use of metal nanocarriers (such as gold nanoparticles or manganese dioxide nanosheets), resulting in uncontrollable surface probe modification and unsatisfactory biosafety. There are few reports on amplified imaging of DNA repair enzyme activity in living cells based on intraCHA. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention provides a method for detecting 8-OG DNA glycosylase activity mediated by an intramolecular catalytic hairpin assembly (RI-intraCHA) nanosystem. In the presence of 8-OG DNA glycosylase, the 8-OG damaged bases within the RI-intraCHA nanosystem are removed, destabilizing the double-stranded DNA RT and releasing the trigger strand T. The released T then initiates the intraCHA reaction cycle, distancing Cy5 and BHQ2, restoring Cy5 fluorescence, and generating an amplified fluorescent signal. This method not only enables sensitive and selective detection of extracellular 8-OG DNA glycosylase activity but also prevents false-positive signals caused by nuclease degradation, facilitating accurate imaging within living cells.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] The first aspect of the present invention provides an intramolecular catalytic hairpin self-assembly nanosystem, comprising: a DNA tetrahedron, wherein three vertices of the DNA tetrahedron are respectively connected to double-stranded DNA, a hairpin probe H1, and a hairpin probe H2;

[0008] The double-stranded DNA is formed by hybridization of an 8-OG-containing recognition strand and a trigger strand; the nucleotide sequence of the trigger strand is shown in SEQ ID NO. 8; the 8-OG-containing recognition strand is obtained by replacing the guanine at positions 7 and 14 with 8-oxidized guanine on the basis of strand R'; the nucleotide sequence of strand R' is shown in SEQ ID NO. 7;

[0009] The nucleotide sequence of the hairpin probe H1 is shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the hairpin probe H2 is shown in SEQ ID NO.2.

[0011] A second aspect of the present invention provides a method for detecting 8-OG DNA glycosylase activity in an extracellular target mediated by an intramolecular catalytic hairpin self-assembly nanosystem, comprising the following steps:

[0012] 1) Construction of intramolecular catalytic hairpin self-assembly nanosystem;

[0013] (2) co-incubating the extracellular target and the intramolecular catalytic hairpin self-assembled nanosystem;

[0014] (3) Perform fluorescence spectrum measurement.

[0015] The third aspect of the present invention provides a method for detecting 8-OG DNA glycosylase activity in cells mediated by an intramolecular catalytic hairpin self-assembly nanosystem, comprising the following steps:

[0016] 1) Construction of intramolecular catalytic hairpin self-assembly nanosystem;

[0017] (2) co-incubating the target cells and the intramolecular catalytic hairpin self-assembly nanosystem;

[0018] (3) Perform laser scanning confocal microscopy imaging.

[0019] One or more embodiments of the present invention have at least the following beneficial effects:

[0020] The present invention provides a method for detecting 8-OG DNA glycosylase activity mediated by an intramolecular catalytic hairpin self-assembly (RI-interCHA) nanosystem. By connecting a double-stranded DNA (including an 8-OG site and a trigger chain) and two hairpin probes to different vertices of a DNA tetrahedron, an RI-intraCHA nanosystem is constructed. When the target 8-OG DNA glycosylase is present, the 8-OG damaged base in the RI-intraCHA nanosystem is removed, causing the double-stranded DNA RT to become unstable and releasing the trigger chain T. Subsequently, the released T can initiate the intraCHA reaction cycle, causing Cy5 and BHQ2 to move away, restoring Cy5 fluorescence, and generating an amplified fluorescent signal. Using this method, sensitive and selective detection of extracellular 8-OG DNA glycosylase activity is achieved, and the detection limit of the RI-intraCHA nanosystem for 8-OG DNA glycosylase is 0.2443 U / mL. Secondly, for intracellular imaging, the RI-intraCHA nanosystem can autonomously enter living cells through endocytosis, where the 8-OG can then be removed by endogenous 8-OG DNA glycosylase, initiating the intraCHA reaction cycle and generating an amplified fluorescent signal. In addition, testing has shown that the RI-interCHA nanosystem cannot be degraded by intracellular nucleases to produce false-positive signals. Therefore, the nanosystem has high stability and specificity in the cellular environment, which is conducive to accurate imaging in living cells. In summary, the RI-interCHA nanosystem is capable of amplifying imaging of 8-OG DNA glycosylase activity in living cells, with the advantages of satisfactory biosafety, self-delivery, high biostability and specificity. It provides a promising tool for studying the basic biology of intracellular DNA repair enzymes and their clinical relevance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1Schematic diagram of the RI-intraCHA nanosystem for detecting 8-OG DNA glycosylase activity (A) and the RI-intraCHA nanosystem for amplified imaging of 8-OG DNA glycosylase activity in living cells (B);

[0023] Figure 2 Agarose gel electrophoresis detection diagram; wherein, A is agarose gel electrophoresis (2.5%) characterizing the preparation of DNA tetrahedron; B is agarose gel electrophoresis (3.5%) characterizing the preparation of DNA double-stranded RT;

[0024] Figure 3 Figures 1 and 2 show agarose gel electrophoresis, AFM, and fluorescence spectra of the RI-intraCHA nanosystem. Figure A shows an agarose gel electrophoresis (2.5%) image of the RI-intraCHA nanosystem. Figure B shows an AFM image of the RI-intraCHA nanosystem. Figure C shows the fluorescence spectra of the RI-intraCHA nanosystem in the presence and absence of the target 8-OG DNA glycosylase (100 U / mL).

[0025] Figure 4 Figures 2 and 3 show the fluorescence spectra, stability, and selectivity of the RI-intraCHA nanosystem. Figure A shows the fluorescence spectra of the RI-intraCHA nanosystem in response to different concentrations of 8-OG DNA glycosylase. Figure B shows the relationship between the ΔF value and the concentration of the target 8-OG DNA glycosylase (the inset shows a linear relationship). ΔF refers to the change in the fluorescence intensity of Cy5 at 662 nm in the presence and absence of the target. Figure C shows the stability and selectivity of the RI-intraCHA nanosystem. The concentrations of the target, DNaseI, UDG, and APE1 were all 100 U / mL. Error bars are standard deviations (SD) (n=3).

[0026] Figure 5 The survival rate of L0-2 cells after incubation with different concentrations of RI-intraCHA nanosystem (50 nM, 100 nM, 150 nM, 200 nM) for 24 h, the error bars are SD (n = 3);

[0027] Figure 6 CLSM images of MCF-7 cells after incubation with free CHA system (RT, H1, and H2, each probe concentration is 100 nM), RI-intraCHA nanosystem (100 nM), or control RI-intraCHA nanosystem (without 8-OG, 100 nM) for 2 h. Scale bar: 20 μm. DETAILED DESCRIPTION

[0028] As described in the background, intramolecular catalytic hairpin assembly (intraCHA) has recently been developed for intracellular imaging. However, non-nucleic acid-activated intraCHA is rarely reported. The only protein-activated intraCHA relies on binding between a protein and its aptamer, but this binding is weak, often resulting in limited initiation efficiency. Furthermore, amplified imaging of DNA repair enzyme activity in living cells requires the use of metal nanocarriers (such as gold nanoparticles or manganese dioxide nanosheets), resulting in uncontrollable surface probe modification and unsatisfactory biosafety.

[0029] In order to solve the above technical problems, the first aspect of the present invention provides an intramolecular catalytic hairpin self-assembly nanosystem, comprising: a DNA tetrahedron, wherein the three vertices of the DNA tetrahedron are respectively connected to double-stranded DNA, a hairpin probe H1, and a hairpin probe H2;

[0030] The double-stranded DNA is formed by hybridization of an 8-OG-containing recognition strand and a trigger strand; the nucleotide sequence of the trigger strand is shown in SEQ ID NO. 8; the 8-OG-containing recognition strand is obtained by replacing the guanine at positions 7 and 14 with 8-oxidized guanine on the basis of strand R'; the nucleotide sequence of strand R' is shown in SEQ ID NO. 7;

[0031] The nucleotide sequence of the hairpin probe H1 is shown in SEQ ID NO. 1;

[0032] The nucleotide sequence of the hairpin probe H2 is shown in SEQ ID NO.2.

[0033] The intramolecular catalytic hairpin self-assembly RI-interCHA nanosystem provided by the present invention has the neck of the hairpin probe H2 labeled with a fluorophore Cy5 and a quencher BHQ2. Initially, the distance between the fluorophore Cy5 and the quencher BHQ2 is relatively close, and no FRET occurs. 8-Oxidized guanine (8-OG) DNA glycosylase was selected as a model DNA repair enzyme, which can recognize and remove 8-OG damaged bases in double-stranded DNA, generating single nucleotide gaps. Under the action of 8-OG DNA glycosylase, 8-OG in double-stranded DNA is removed, causing the double-stranded DNA RT to become unstable and releasing the trigger chain T. Subsequently, the released T can initiate the intraCHA reaction cycle, causing Cy5 and BHQ2 to move away, and the Cy5 fluorescence to recover, resulting in efficient FRET.

[0034] The construction of the RI-interCHA nanosystem can sensitively and selectively detect extracellular 8-OG DNA glycosylase activity. More importantly, based on the FRET signal output mode, the RI-interCHA nanosystem cannot be degraded by nucleases in the cell to produce false positive signals, thereby improving the detection accuracy and having high stability and specificity. In addition, DNA tetrahedron, as a typical FNA, has the advantages of cell permeability and biostability. With the cell permeability of DNA tetrahedron, the RI-intraCHA nanosystem can autonomously enter living cells and accurately and in situ image the activity of 8-OG DNA glycosylase in the cell. In general, the RI-interCHA nanosystem can amplify and image the activity of 8-OG DNA glycosylase in living cells, and has the advantages of satisfactory biosafety, self-delivery, high biostability and specificity.

[0035] Furthermore, the preparation method of the intramolecular catalytic hairpin self-assembly nanosystem comprises the following steps:

[0036] 1) Preparation of DNA tetrahedrons: Mix four single-stranded DNA strands at equimolar concentrations and incubate at 75-85°C for 1-3 minutes, then at 55-65°C for 1-3 minutes, and finally store at 3-5°C.

[0037] 2) Preparation of double-stranded DNA RT: Mix equimolar amounts of the recognition strand R containing 8-OG bases and the trigger strand T, incubate at 90-100°C for 5-15 minutes, and cool naturally to 20-30°C;

[0038] 3) Preparing DNA tetrahedrons connected to double-stranded DNA RT: mixing equimolar amounts of the DNA tetrahedrons prepared in step 1) and the double-stranded DNA RT prepared in step 2), and incubating at 20-30° C. for 0.5-1.5 h;

[0039] 4) Preparation of hairpin probes: Incubate hairpin probe H1 and hairpin probe H2 at 85-95°C for 3-8 minutes, and then cool naturally to 20-30°C;

[0040] 5) Preparation of intramolecular catalytic hairpin self-assembly nanosystem: Equimolar amounts of hairpin probe H1, hairpin probe H2, and DNA tetrahedron connected to double-stranded DNA RT were mixed and incubated at 20-30° C. for 0.5-1.5 h.

[0041] The processes from step 1) to step 5) are all carried out in 1×PBS buffer solution.

[0042] The second aspect of the present invention provides a method for detecting 8-OG DNA glycosylase activity in extracellular targets mediated by an intramolecular catalytic hairpin self-assembly nanosystem, specifically:

[0043] 1) Construction of intramolecular catalytic hairpin self-assembly nanosystem;

[0044] (2) co-incubating the extracellular target and the intramolecular catalytic hairpin self-assembled nanosystem;

[0045] (3) Perform fluorescence spectrum measurement.

[0046] In some examples of this embodiment, the incubation is at 37° C. for 40-80 min, preferably 60 min.

[0047] In some examples of this embodiment, during the fluorescence spectrum measurement, the excitation wavelength is 635 nm, the emission wavelength is 655 nm-750 nm; the excitation and emission slit widths are 10 nm, and the photomultiplier tube voltage is 700 V.

[0048] The third aspect of the present invention provides a method for detecting 8-OG DNA glycosylase activity in cells mediated by an intramolecular catalytic hairpin self-assembly nanosystem, specifically:

[0049] 1) Construction of intramolecular catalytic hairpin self-assembly nanosystem;

[0050] (2) co-incubating the target cells and the intramolecular catalytic hairpin self-assembly nanosystem;

[0051] (3) Perform laser scanning confocal microscopy imaging.

[0052] In some examples of this embodiment, the target cells are seeded on a confocal culture dish, incubated at 37° C. for 20-25 hours, and then co-incubated with the intramolecular catalytic hairpin self-assembly nanosystem.

[0053] In some examples of this embodiment, the co-incubation is: incubation at 35-40° C. for 1-3 hours.

[0054] Furthermore, the co-incubation is: incubation at 37° C. for 2 hours.

[0055] In some examples of this embodiment, the laser scanning confocal microscope imaging is: under the excitation light of 633nm wavelength, collecting Cy5 emission light in the range of 650nm-700nm.

[0056] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0057] Example 1

[0058] 1. Experimental part

[0059] (1) Materials and instruments

[0060] All DNA oligonucleotides (Table 1) and 10× PBS buffer (1.37 M sodium chloride, 26.83 mM potassium chloride, 81 mM sodium dihydrogen phosphate, 17.6 mM potassium dihydrogen phosphate) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). 8-OG DNA glycosylase (Fpg), uracil DNA glycosylase (UDG), apurinic / apyrimidinic endonuclease 1 (APE1), and DNase I were provided by New England Biolabs (Beijing, China). MTT and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Missouri, USA). Gel images were acquired using a 4600SF gel imaging system (Tianneng, China). Atomic force microscopy (AFM) images were acquired using a Bruker microscope (Bruker, Germany). Fluorescence spectra were acquired using an F-7000 fluorescence spectrophotometer (Hitachi, Japan). Cell viability was determined using a Spark microplate reader (Tecan, Switzerland). Cell imaging was performed using a LSM-880 confocal laser scanning microscope (CLSM) (Zeiss, Germany).

[0061] (2) Preparation of RI-intraCHA nanosystem

[0062] First, DNA tetrahedrons were prepared by mixing four single-stranded DNA strands (A, B, C, and D) at equimolar concentrations in 1× PBS buffer, incubating at 80°C for 2 minutes, then at 60°C for another 2 minutes, and finally storing at 4°C to obtain DNA tetrahedrons.

[0063] Second, prepare double-stranded DNA RT. Equimolar amounts of the 8-OG-containing recognition strand R and trigger strand T were mixed and incubated at 95°C for 10 minutes. The mixture was then cooled to 25°C to obtain double-stranded DNA RT. The 8-OG-containing recognition strand R was prepared by replacing the guanine at positions 7 and 14 with 8-oxidized guanine based on strand R' in Table 1.

[0064] Third, DNA tetrahedrons connected to double-stranded DNA RT were prepared. Equimolar amounts of the double-stranded DNA RT and DNA tetrahedrons were mixed and incubated at 25° C. for 1 hour to obtain DNA tetrahedrons connected to double-stranded DNA RT.

[0065] Fourth, prepare the hairpin probe. To obtain the hairpin structure, incubate H1 and H2 at 90°C for 5 minutes, then cool naturally to 25°C.

[0066] Fifth, prepare the RI-intraCHA nanosystem. Equimolar amounts of H1, H2, and DNA tetrahedrons linked to double-stranded DNA RT were mixed and incubated at 25°C for 1 hour to produce the RI-intraCHA nanosystem. The prepared RI-intraCHA nanosystem was stored at 4°C for subsequent use.

[0067] Table 1. DNA oligonucleotide sequences used in this experiment.

[0068]

[0069]

[0070] (3) Gel electrophoresis

[0071] Each assembly step during the preparation of the RI-intraCHA nanosystem was characterized by agarose gel electrophoresis. 10 μL of sample was mixed with 2 μL of 6× loading buffer and loaded onto an agarose gel containing GelRed. The gel was then electrophoresed in 1× TBE buffer at 100 V for 1 hour. Finally, the gel was imaged.

[0072] (4) Fluorescence detection

[0073] To verify the RI-intraCHA nanosystem's detection response to targets in buffered solution, various concentrations of 8-OG DNA glycosylase (100 μL, 100 nM) were added to the RI-intraCHA nanosystem. After incubation at 37°C for 1 hour, fluorescence spectra were collected from 655 to 750 nm under an excitation wavelength of 635 nm. The excitation and emission slit widths were 10 nm, and the photomultiplier tube voltage was 700 V.

[0074] (5) Intracellular imaging of 8-OG DNA glycosylase activity

[0075] The RI-intraCHA nanoparticle system was used to detect 8-OG DNA glycosylase activity in breast cancer (MCF-7) cells. The cells were seeded into confocal microplates and incubated at 37°C for 24 hours, after which the culture medium was removed. Fresh serum-free culture medium containing 100 nM RI-intraCHA nanoparticle system was then added and incubated at 37°C for 2 hours. Finally, after three washes with PBS buffer, the cells were imaged using CLSM, with Cy5 emission light collected in the 650-700 nm range under excitation at 633 nm.

[0076] (6) Cell survival rate

[0077] Normal human hepatocytes (L0-2) were seeded in 96-well plates and incubated at 37°C for 24 hours, after which the culture medium was removed. Fresh serum-free culture medium containing varying concentrations of the RI-intraCHA nanosystem (50 nM, 100 nM, 150 nM, and 200 nM) was then added to each well and incubated at 37°C for 24 hours. Subsequently, the cells in each well were mixed with 20 μL of MTT (5 mg / mL) and incubated at 37°C for 4 hours. Finally, 150 μL of dimethyl sulfoxide (DMSO) was added, and the absorbance of the samples was measured at 490 nm.

[0078] 2. Results and Discussion

[0079] (1) Principle of RI-intraCHA nanosystem for 8-OG DNA glycosylase activity detection

[0080] The principle of RI-intraCHA nanosystem for 8-OG DNA glycosylase activity detection is as follows Figure 1 The RI-intraCHA nanosystem consists of a DNA tetrahedron, a double-stranded DNA RT, and two hairpin probes, H1 and H2. The double-stranded DNA RT is formed by hybridizing an 8-OG-containing recognition strand (R) with a trigger strand (T); the neck of hairpin H2 is labeled with the fluorophore Cy5 and the quencher BHQ2. To construct the RI-intraCHA nanosystem, the double-stranded DNA RT, H1, and H2 are attached to the three vertices of the DNA tetrahedron through complementary hybridization.

[0081] When the target, 8-OG DNA glycosylase, is present, the 8-OG damaged bases in the RI-intraCHA nanosystem are removed, causing double-stranded DNA RT to become unstable and releasing the trigger strand T. The released T then initiates the intraCHA reaction cycle, distancing Cy5 and BHQ2, restoring Cy5 fluorescence, and generating an amplified fluorescent signal. This fluorescent signal can indicate the activity level of 8-OG DNA glycosylase. Furthermore, for intracellular imaging, the RI-intraCHA nanosystem can enter cells via endocytosis, where the 8-OG is then removed by endogenous 8-OG DNA glycosylase, initiating the intraCHA reaction cycle and generating an amplified fluorescent signal. Therefore, the constructed RI-intraCHA nanosystem provides a platform for amplified imaging of 8-OG DNA glycosylase activity in living cells.

[0082] (2) Characterization of RI-intraCHA nanosystem and study of its detection feasibility

[0083] First, the formation of DNA tetrahedrons was studied by agarose gel electrophoresis. Figure 2As shown in Figure A, as chains A, B, C, and D are gradually added, the mobility of the mixture gradually decreases, which is attributed to the increase in the molecular weight of the hybridization product, indicating the formation of DNA tetrahedrons. Subsequently, the formation of double-stranded DNA RT was also studied by agarose gel electrophoresis. Figure 2 As shown in B, the study found that the migration rate of the mixture of R and T was lower than that of single-stranded DNA R or T. Finally, the assembly of DNA tetrahedron with RT, H1, and H2 was studied by agarose gel electrophoresis. Figure 3 Bands 1-4 in A, the mixture containing DNA tetrahedron, RT, H1 and H2 has the slowest migration, indicating that RT, H1 and H2 are successfully connected with DNA tetrahedron to form RI-intraCHA nanosystem. In addition, the RI-intraCHA nanosystem was directly characterized by AFM, e.g. Figure 3 As shown in B.

[0084] To demonstrate the feasibility of the detection, the fluorescence response of the RI-intraCHA nanosystem to 8-OG DNA glycosylase was tested. Figure 3 As shown in Figure C, the fluorescence intensity of Cy5 increased significantly in the presence of 8-OG DNA glycosylase. These results indicate that the designed RI-intraCHA nanosystem can be used for amplified detection of 8-OG DNA glycosylase activity.

[0085] (3) Performance of RI-intraCHA nanosystem

[0086] In order to investigate the activation efficiency and detection sensitivity of the RI-intraCHA nanosystem, different concentrations of 8-OG DNA glycosylase were added to the nanosystem. Figure 4 As shown in A, when the concentration of the target 8-OG DNA glycosylase increases, the fluorescence intensity of Cy5 gradually increases. Figure 4 As shown in Figure B, the ΔF value is proportional to the target concentration within the target concentration range of 0 U / mL to 10 U / mL. The limit of detection (LOD) is 0.2443 U / mL (3σ / slope). This indicates that the intraCHA reaction in the RI-intraCHA nanosystem is effectively initiated by the target 8-OG DNA glycosylase, demonstrating good sensitivity to the target.

[0087] Subsequently, the stability and selectivity of the RI-intraCHA nanosystem were investigated. Figure 4As shown in Figure C, only the target compound can induce high fluorescence intensity in the nanosystem. In contrast, DNase I only causes the nanosystem to exhibit low fluorescence intensity, indicating that the nanosystem is resistant to nuclease degradation. Furthermore, other interferases, UDG or APE1, also only cause the nanosystem to exhibit weak fluorescence intensity, as these enzymes are unable to react with the 8-OG site in the nanosystem. These results demonstrate the excellent stability and selectivity of the nanosystem.

[0088] (4) RI-intraCHA nanosystem for imaging 8-OG DNA glycosylase activity in living cells

[0089] The cytotoxicity of the RI-intraCHA nanosystem was evaluated using the MTT assay. Figure 5 As shown in the figure, after incubation with different concentrations of RI-intraCHA (50nM, 100nM, 150nM, 200nM) for 24h, L0-2 cells retained more than 90% of their viability, indicating that the designed RI-intraCHA nanosystem is safe for cells.

[0090] Subsequently, the RI-intraCHA nanosystem was used to amplify and image the activity of 8-OG DNA glycosylase in living cells. Figure 6 As shown, bright Cy5 fluorescence was detected after 2 hours of incubation of MCF-7 cells with the RI-intraCHA nanosystem. This demonstrates that, in the absence of metal nanocarriers or transfection agents, the nanosystem can autonomously enter living cells and react with endogenous 8-OG DNA glycosylase, effectively initiating the intraCHA reaction and generating significant fluorescence. On the other hand, no significant fluorescence was detected when MCF-7 cells were incubated with the free CHA systems (RT, H1, and H2), indicating that the free CHA systems cannot autonomously enter living cells. This also confirms that the DNA tetrahedron is essential for enhancing the cell permeability of the RI-intraCHA nanosystem. Furthermore, when a control RI-intraCHA nanosystem (using guanine instead of 8-OG) was introduced into MCF-7 cells, negligible fluorescence was generated. This indicates that the nanosystem is not degraded by intracellular nucleases, which could produce false-positive signals, confirming the high stability and specificity of the nanosystem in the cellular environment. In summary, the RI-interCHA nanosystem is capable of amplifying and imaging 8-OG DNA glycosylase activity in living cells, and has the advantages of satisfactory biosafety, self-delivery, high biostability and specificity.

[0091] In summary, the present invention has developed an RI-intraCHA nanosystem, and its operation can be used to amplify and image the activity of DNA repair enzymes in living cells. 8-OG DNA glycosylase was selected as a model DNA repair enzyme. By connecting double-stranded DNA RT and hairpin probes H1 and H2 at the three vertices of a DNA tetrahedron, a highly integrated RI-intraCHA nanosystem was constructed, which can autonomously enter living cells. After the 8-OG damage in the system is repaired by 8-OG DNA glycosylase, the intraCHA reaction cycle is efficiently initiated, showing good sensitivity to the target enzyme. In addition, the RI-intraCHA nanosystem also has satisfactory biosafety and biostability. Furthermore, the RI-intraCHA nanosystem can be used as a signal amplifier in living cells to achieve amplified imaging of endogenous 8-OG DNA glycosylase activity. The designed RI-intraCHA nanosystem provides a promising tool for studying the basic biology of intracellular DNA repair enzymes and their clinical relevance.

[0092] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An intramolecular catalytic hairpin self-assembly nanosystem, characterized in that: include: DNA tetrahedron, with double-stranded DNA, hairpin probe H1, and hairpin probe H2 connected to the three vertices of the DNA tetrahedron respectively; The double-stranded DNA is formed by hybridization of an 8-OG-containing recognition strand and a trigger strand; the nucleotide sequence of the trigger strand is shown in SEQ ID NO. 8; the 8-OG-containing recognition strand is obtained by replacing the guanine at positions 7 and 14 with 8-oxidized guanine on the basis of strand R'; the nucleotide sequence of strand R' is shown in SEQ ID NO. 7; The nucleotide sequence of the hairpin probe H1 is shown in SEQ ID NO.1; The nucleotide sequence of the hairpin probe H2 is shown in SEQ ID NO.

2.

2. A method for preparing the intramolecular catalytic hairpin self-assembly nanosystem according to claim 1, characterized in that: The following steps are involved: 1) Preparation of DNA tetrahedrons: Mix four single-stranded DNA strands at equimolar concentrations and incubate at 75-85°C for 1-3 min, then at 55-65°C for 1-3 min, and finally store at 3-5°C. 2) Prepare double-stranded DNA RT: Mix equimolar amounts of the recognition strand R containing 8-OG bases and the trigger strand T, incubate at 90-100°C for 5-15 minutes, and cool naturally to 20-30°C. 3) Preparation of DNA tetrahedrons linked to double-stranded DNA RT: Mix equimolar amounts of the DNA tetrahedrons prepared in step 1) and the double-stranded DNA RT prepared in step 2) and incubate at 20-30°C for 0.5-1.5 h; 4) Preparation of hairpin probes: Incubate hairpin probes H1 and H2 at 85-95°C for 3-8 minutes, then cool to 20-30°C. 5) Preparation of intramolecular catalytic hairpin self-assembly nanosystem: Equimolar amounts of hairpin probe H1, hairpin probe H2, and DNA tetrahedron connected to double-stranded DNA RT were mixed and incubated at 20-30°C for 0.5-1.5 h.

3. The preparation method according to claim 2, wherein The processes from step 1) to step 5) were all carried out in 1× PBS buffer solution.

4. A method for detecting 8-OG DNA glycosylase activity in extracellular targets mediated by the intramolecular catalytic hairpin self-assembly nanosystem according to claim 1, characterized in that: The steps include: 1) Construction of intramolecular catalytic hairpin self-assembly nanosystems; (2) co-incubating the extracellular target and the intramolecular catalytic hairpin self-assembled nanosystem; (3) Perform fluorescence spectrum measurement.

5. The method according to claim 4, wherein Incubate at 37°C for 40-80 min.

6. The method according to claim 5, wherein Incubate at 37°C for 60 min.

7. The method according to claim 4, wherein During the fluorescence spectrum measurement, the excitation wavelength was 635 nm, the emission wavelength was 655 nm-750 nm, the excitation and emission slit widths were 10 nm, and the photomultiplier tube voltage was 700 V.

8. A method for detecting 8-OG DNA glycosylase activity in cells mediated by the intramolecular catalytic hairpin self-assembly nanosystem according to claim 1, characterized in that: The steps include: 1) Construction of intramolecular catalytic hairpin self-assembly nanosystems; (2) co-incubating the target cells and the intramolecular catalytic hairpin self-assembly nanosystem; (3) Perform laser scanning confocal microscopy imaging.

9. The method according to claim 8, wherein The target cells were seeded on a confocal culture dish, incubated at 37°C for 20-25 h, and then co-incubated with the intramolecular catalytic hairpin self-assembly nanosystem.

10. The method according to claim 8, wherein The co-incubation is: incubating at 35-40° C. for 1-3 h.

11. The method according to claim 10, wherein The co-incubation is: incubating at 37° C. for 2 h.

12. The method according to claim 8, wherein The laser scanning confocal microscope imaging is as follows: under the excitation light of 633 nm wavelength, the Cy5 emission light within the range of 650 nm-700 nm is collected.

Images