Intelligent catalytic DNA hydrogel as well as preparation method and application thereof

Abstract

The invention discloses intelligent catalytic DNA hydrogel and a preparation method and application thereof, the preparation method comprises the following steps: S1, two annular DNA template chains are provided, the annular DNA template chain 1 comprises a nucleic acid aptamer sequence of a specific targeting bacterium surface component, and the annular DNA template chain 2 comprises a sequence capable of forming a G-quadruplex; s2, extending the annular DNA template chain through a rolling circle amplification reaction to form DNA hydrogel with a three-dimensional cross-linked network structure; and S3, mixing the DNA hydrogel with potassium ions and hemin, so that the G-quadruplex sequence is combined with the hemin to form the composite catalysis unit with peroxidase-like activity. The DNA hydrogel integrates multiple functions of accurate targeting, intelligent catalysis, photothermal effect and the like, the killing effect of drug-resistant bacteria is remarkably improved through a three-level joint control mechanism, an innovative solution is provided for treatment of drug-resistant bacterial infection, and the DNA hydrogel has important clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, particularly to nucleic acids, and specifically to an intelligent catalytic DNA hydrogel and its preparation method and application. Background Art

[0002] Drug-resistant bacterial infections have become a major threat to global public health. Drug-resistant bacteria refer to those bacteria that can resist the action of antibacterial drugs through various mechanisms, thus avoiding the risk of being killed. With the widespread use of antibacterial drugs, the emergence and spread of drug-resistant bacteria far exceed the R & D speed of new antibacterial drugs, resulting in a significant decline in the efficacy of traditional antibiotics. Currently, almost all types of bacteria have developed drug resistance globally, increasing the treatment difficulty of drug-resistant bacterial infections, rising the fatality rate, and significantly increasing medical costs.

[0003] Existing antibacterial treatment strategies, such as the photothermal effect, although having characteristics such as minimally invasive, long-acting, and safe, still face challenges in the treatment of drug-resistant bacterial infections. The photothermal effect kills cancer cells by converting light energy into heat energy, but it has a problem of poor targeting in the treatment of drug-resistant bacterial infections. In addition, the high temperature of the photothermal effect may cause damage to normal tissues, limiting its further development in clinical applications.

[0004] Therefore, it is necessary to improve the deficiencies in the existing technology to solve the above problems. Summary of the Invention

[0005] The present invention overcomes the deficiencies of the existing technology and provides an intelligent catalytic DNA hydrogel and its preparation method and application. Through a three-level control mechanism of precise targeting, intelligent catalysis, and the photothermal effect, efficient killing of drug-resistant bacteria is achieved.

[0006] To achieve the above object, the technical solution adopted by the present invention is: A preparation method of an intelligent catalytic DNA hydrogel, comprising the following steps:

[0007] S1. Provide two circular DNA template strands, wherein circular DNA template strand 1 contains a nucleic acid aptamer sequence specifically targeting bacterial surface components, and circular DNA template strand 2 contains a sequence capable of forming a G-quadruplex;

[0008] S2. Extend the circular DNA template strands through a rolling circle amplification reaction to form a DNA hydrogel with a three-dimensional cross-linked network structure;

[0009] S3. Mix the DNA hydrogel with potassium ions and hemin so that the G-quadruplex sequence binds to hemin to form a composite catalytic unit with peroxidase-like activity.

[0010] In a preferred embodiment of the present invention, in the step of S1, the sequence of the DNA template strand 1 is as shown in SEQ ID NO: 1, and the sequence of the DNA template strand 2 is as shown in SEQ ID NO: 2.

[0011] In a preferred embodiment of the present invention, in the step of S1, the nucleic acid aptamer sequence specifically targets bacterial peptidoglycan, and the polymerase used in the rolling circle amplification reaction is a strand displacement DNA polymerase.

[0012] In a preferred embodiment of the present invention, in the step of S1, the circular DNA template strand includes the following steps:

[0013] S11. Phosphorylate the linear DNA template strand;

[0014] S12. Anneal the phosphorylated linear DNA template with a complementary primer and catalyze the formation of a circular structure through a ligase.

[0015] In a preferred embodiment of the present invention, in the step of S11, the phosphorylation treatment is carried out in a reaction system containing ATP; in the step of S12, the ligase is T4 DNA ligase.

[0016] In a preferred embodiment of the present invention, in the step of S3, the concentration of potassium ions is 100 - 200 mM, and the concentration of hemin is 40 - 100 μM.

[0017] The present invention provides an intelligent catalytic DNA hydrogel prepared by the preparation method described in any one of the foregoing, and the DNA hydrogel includes:

[0018] A nucleic acid aptamer functional module for specifically capturing target bacteria;

[0019] A G-quadruplex / hemin composite catalytic unit capable of catalyzing the in-situ polymerization of dopamine to form a polydopamine coating layer in an acidic microenvironment;

[0020] The microstructure of the DNA hydrogel is a reticular void structure.

[0021] In a preferred embodiment of the present invention, the polydopamine coating layer generates a photothermal effect under near-infrared light irradiation.

[0022] The present invention provides an application of the intelligent catalytic DNA hydrogel described in any one of the foregoing in the preparation of a drug for treating drug-resistant bacterial infections.

[0023] In a preferred embodiment of the present invention, the drug exerts its effect through the following mechanism:

[0024] Utilize nucleic acid aptamers to target and capture drug-resistant bacteria;

[0025] In the bacterial infection microenvironment, the composite catalytic unit catalyzes the polymerization of dopamine to form a polydopamine coating layer;

[0026] The polydopamine coating layer is excited by near-infrared light to generate a photothermal effect, killing drug-resistant bacteria.

[0027] The present invention solves the defects existing in the background technology and has the following beneficial effects:

[0028] (1) The present invention provides an intelligent catalytic DNA hydrogel, its preparation method and application. Through the nucleic acid aptamer (Apt), precise targeting capture of drug-resistant bacteria is achieved, and combined with the in-situ catalytic function of the G4 / Hemin composite structure, the accuracy and effectiveness of treatment are significantly improved.

[0029] (2) In the present invention, the designed DNA hydrogel system can be specifically activated in the infection microenvironment, reducing damage to normal tissues and improving the safety of treatment.

[0030] (3) In the present invention, the designed DNA hydrogel integrates multiple functions such as precise targeting, intelligent catalysis, and photothermal effect. Through a three-level control mechanism, the killing effect of drug-resistant bacteria is significantly improved, providing an innovative solution for the treatment of drug-resistant bacterial infections and having important clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts;

[0032] Figure 1 is the schematic diagram of the preparation of the DNA hydrogel of the present invention;

[0033] Figure 2 is the microscopic structure diagram of the DNA hydrogel prepared by the present invention under a cryogenic scanning electron microscope (Cryo-SEM);

[0034] Figure 3 is the circular dichroism spectrum diagram for verifying the loading of Apt (A) and G4 (B) of the present invention;

[0035] Figure 4 is the performance effect diagram of the in vitro catalysis of DA to form PDA by the DNA hydrogel of the present invention. Among them, Figure 4 A is the component diagram of groups ①-⑥, Figure 4 B is the absorbance performance diagram of groups ①-⑥,Figure 4 C is the photothermal performance diagram of groups ①-⑥;

[0036] Figure 5 It is the micrograph of the surface of drug-resistant bacteria wrapped with a PDA coating layer under a transmission electron microscope (TEM) of the present invention. Among them, Figure 5 A is the micrograph of the surface of drug-resistant bacteria wrapped with a PDA coating layer in Example 4, Figure 5 B is the micrograph of the surface of drug-resistant bacteria wrapped with a PDA coating layer in Comparative Example 1;

[0037] Figure 6 It is the laser scanning confocal microscope image for verifying the Apt targeting ability in the DNA hydrogel of the present invention. Among them, Figure 6 A is the laser scanning confocal microscope image of the Apt targeting ability in the DNA hydrogel of Example 5, Figure 6 B is the laser scanning confocal microscope image of the Apt targeting ability in the DNA hydrogel of Comparative Example 2;

[0038] Figure 7 It is the antibacterial culture plate of the DNA hydrogel of the present invention. Detailed implementation manners

[0039] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0040] Many specific details are set forth in the following description in order to provide a thorough understanding of the present invention. However, the present invention may be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited by the specific embodiments disclosed below.

[0041] Application overview:

[0042] Due to its unique programmability, biocompatibility, and functional modular integration ability, DNA hydrogel has always been regarded as a potential carrier for drug delivery and antibacterial therapy. Such hydrogels can load and slowly release a variety of antibacterial components, such as chemotherapeutic drugs, antimicrobial peptides, and metal nanoparticles, thus showing great application potential in antibacterial therapy. However, in in-depth research and practical applications, it has gradually been found that there are significant limitations in the existing DNA hydrogel system.

[0043] Most existing DNA hydrogel systems mainly rely on a single bactericidal mechanism, greatly limiting their antibacterial effects and scope of application. More critically, these systems often struggle to precisely activate their functions in complex infection microenvironments, resulting in a significant reduction in treatment efficacy. The infection microenvironment typically has unique biochemical characteristics, such as specific pH values, ion concentrations, and the presence of specific biomolecules, etc., while existing DNA hydrogels often fail to fully adapt to and utilize these characteristics to achieve more precise and efficient treatment.

[0044] In view of the above findings, the concept of this invention is to propose an intelligent catalytic DNA hydrogel and its preparation method and application, which can achieve precise capture of drug-resistant bacteria through precise targeting technology, while using an intelligent catalytic mechanism to specifically activate its bactericidal function in the infection microenvironment, and combining with the photothermal effect to further enhance the bactericidal effect, reduce damage to normal tissues, and improve the safety and effectiveness of treatment.

[0045] It should be noted that the raw materials, equipment, reagents, etc. used in this invention can all be obtained through market purchase or by means of existing technology preparation.

[0046] The nucleotide sequences used in the following examples are shown in Table 1.

[0047] Table 1 Nucleotide sequences used:

[0048]

[0049]

[0050] A preparation method of an intelligent catalytic DNA hydrogel, comprising the following steps:

[0051] S1. React DNA template strand 1 (Template-1, 10 - 50 μM), T4 PNK buffer (1×), T4 PNK enzyme (100 - 500 U / mL), and ATP (2 - 10 mM) at 37 °C for 2 - 4 h to obtain phosphorylated DNA template strand 1. The treatment of DNA template strand 2 (Template-2) is the same as above;

[0052] S2. Denature phosphorylated DNA template strand 1 (2 - 10 μM), primer strand 1 (Primer-1, 2 - 10 μM), and T4 ligase buffer (1×) at 85 °C for 5 - 10 min. After cooling at room temperature for 10 - 20 min, add T4 DNA ligase (2000 - 50000 U / mL) and incubate at 16 °C for 12 - 16 h. Finally, incubate at 65 °C for 10 - 20 min to inactivate T4 ligase, obtaining circularized DNA template strand 1. The treatment of phosphorylated DNA template strand 2 is the same as above;

[0053] S3. React the circularized DNA template strand 1 (1 - 10 μM), circularized DNA template strand 2 (1 - 10 μM), Phi29 DNA polymerase Buffer (1×), Phi29 DNA polymerase (100 - 200 U / mL), and dNTPs (1 - 10 mM) at 37 °C for 4 - 8 h, and then incubate at 75 °C for 10 - 20 min to inactivate the Phi29 DNA polymerase, thus obtaining the functionalized DNA hydrogel;

[0054] S4. Add potassium ion (K + ) solution (100 - 200 mM) and hemin (40 - 100 μM) dissolved in DMSO to the DNA hydrogel, and incubate at 37 °C for 0.5 - 1 h to obtain G4 / Hemin (a composite structure with peroxidase-like activity).

[0055] The present invention provides an intelligent catalytic DNA hydrogel prepared by the preparation method of any one of the foregoing. The DNA hydrogel includes: a nucleic acid aptamer functional module for specifically capturing target bacteria; a G-quadruplex / hemin composite catalytic unit capable of catalyzing the in-situ polymerization of dopamine to form a polydopamine coating layer in an acidic microenvironment; and the microstructure of the DNA hydrogel is a reticular void structure.

[0056] In some specific embodiments, the polydopamine coating layer generates a photothermal effect under near-infrared light irradiation.

[0057] The present invention provides an application of the intelligent catalytic DNA hydrogel of any one of the foregoing in the preparation of a drug for treating drug-resistant bacterial infections.

[0058] In some specific embodiments, the drug exerts its effect through the following mechanism: using nucleic acid aptamers to target and capture drug-resistant bacteria; in the bacterial infection microenvironment, the composite catalytic unit catalyzes the polymerization of dopamine to form a polydopamine coating layer; and the polydopamine coating layer is excited by near-infrared light to generate a photothermal effect to kill drug-resistant bacteria.

[0059] Example 1

[0060] In this example, as shown in the preparation schematic diagram, the preparation method of the DNA hydrogel includes the following steps: Figure 1

[0061] ​S1. The total reaction volume is 10 μL. Add 2 μL of DNA template strand 1 (50 μM), 1 μL of 10×T4 PNK buffer (70 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT), 0.1 μL of T4 PNK (10000 U / mL), 2 μL of ATP (10 mM), and 4.9 μL of ddH2O, and react at 37 °C for 2 h to obtain phosphorylated DNA template strand 1. The treatment of DNA template strand 2 is the same as above;

[0062] S2. The total reaction volume is 20 μL. Add 4 μL of phosphorylated DNA template strand 1 (10 μM), 2 μL of primer strand 1 (10 μM), and 2 μL of 10×T4 ligase Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT), denature at 85 °C for 5 min, then cool to room temperature for 15 min. Then add 0.1 μL of T4 DNA ligase (400000 U / mL) and 11.9 μL of ddH2O, incubate at 16 °C for 12 h, and then incubate at 65 °C for 20 min to inactivate T4 DNA ligase, obtaining circularized DNA template strand 1. The treatment of phosphorylated DNA template strand 2 is the same as above;

[0063] S3. The total reaction volume is 100 μL. Add 10 μL of circularized DNA template strand 1 (2 μM), 10 μL of circularized DNA template strand 2 (2 μM), 10 μL of 10×Phi29 DNA polymerase Buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT), 1 μL of Phi29 DNA polymerase (10000 U / mL), 10 μL of dNTPs (10 mM), and 59 μL of ddH2O, react at 37 °C for 4 h, and then incubate at 75 °C for 10 min to inactivate Phi29 DNA polymerase, obtaining functionalized DNA hydrogel;

[0064] S4. Add K with a final concentration of 140 mM + and 1 mM of Hemin to the DNA hydrogel, and incubate at 37 °C for 10 min to form G4 / Hemin.

[0065] Example 2

[0066] In this example, it was explored whether the DNA hydrogel was successfully synthesized, and the results are as follows:

[0067] First, the DNA hydrogel was cryo-fixed by high-pressure freezing or liquid nitrogen slush method. Subsequently, the sample was transferred to a vacuum sputtering instrument under low-temperature conditions for fracture treatment to expose the fresh fracture surface of the sample. According to the specific situation of the sample, sublimation treatment was carried out to prevent the surface from being coated with ice, and then the sample was conductively sprayed. After completing the above steps, the sample was placed on the cold stage of a cryogenic scanning electron microscope (Cryo-SEM) through a cryo-transfer system for observation. The results are as Figure 2 shown, and the DNA hydrogel presents a microscopic network structure.

[0068] 50 mM of complementary DNA template strand 1, Apt, complementary DNA template strand 2, and the sample of complementary DNA template strand 2 with 140 mM K + were respectively taken for circular dichroism spectroscopy tests. During the test, the bandwidth of the instrument was set to 2 nm, and the wavelength range was 180 - 320 nm. All samples were measured in parallel three times and then the average value was taken. The results are as Figure 3 shown in A. The positive Cotton effect peaks of complementary DNA template strand 1 and the Apt sequence overlap at 190 nm, indicating the presence of the Apt sequence in complementary DNA template strand 1. In addition, as Figure 3 shown in B, in the presence of 140 mM K + , the positive Cotton effect peak of complementary DNA template strand 2 undergoes a red shift, indicating that complementary DNA template strand 2 forms a G4 structure capable of loading Hemin.

[0069] Example 3

[0070] In this example, the catalytic effect of the DNA hydrogel on DA and its conditions were explored. The steps are as follows:

[0071] There were a total of 6 groups in the experiment. The steps of group ① are as follows. 100 μL of DNA hydrogel, 9.2 μL K + (3 M), and 8 μL of Hemin were incubated at 37 °C for 10 min to form a G4 / Hemin complex structure. Then, 30.8 μL of HEPES buffer solution (pH 6.5), 2 μL of H2O2 (100 mM), and 50 μL of DA (200 mM) were added and reacted at 37 °C for 30 min. The role of the HEPES buffer solution (pH 6.5) and H2O2 was to simulate the acidic microenvironment and endogenous H2O2 in the microenvironment of drug-resistant bacteria infection. The steps of the remaining groups were similar to those of ①, except that there were changes in the corresponding components. There were a total of 6 groups, and the specific components are shown in Figure 4 A.

[0072] The reaction products of each group were put into an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance. As Figure 4As shown in B, Group ① has the largest absorbance, indicating that the presence of DNA hydrogel and H2O2 is necessary for the formation of PDA. Finally, it was irradiated with an 808 nm infrared laser at 1800 mA for 10 min, and the temperature was recorded every 10 s with an infrared thermal imager. The results are as Figure 4 shown in C. The catalytically formed PDA exhibits strong near-infrared (NIR) absorption and can be heated to 60 °C within 3 min, showing excellent photothermal performance.

[0073] Example 4

[0074] In this example, it was explored whether the DNA hydrogel could catalyze the in-situ formation of a PDA coating on the surface of drug-resistant bacteria. The steps are as follows:

[0075] First, the drug-resistant Escherichia coli was pretreated with the materials. 100 μL of DNA hydrogel, 9.2 μL of K + (3 M) and 8 μL of hemin were incubated at 37 °C for 10 min to form a G4 / hemin composite structure. Then, 30.8 μL of HEPES buffer solution (pH 6.5), 2 μL of H2O2 (100 mM), 50 μL of DA (200 mM), and a mung bean-sized drug-resistant Escherichia coli bacterial solution (0.5 - 0.8) centrifuged precipitate were added and reacted at 37 °C for 30 min.

[0076] 10 μL of the pretreated sample was taken and dropped onto a copper grid, allowed to precipitate for 1 min, and then the floating liquid was blotted with filter paper. Next, 10 μL of uranyl acetate was dropped onto the copper grid, allowed to precipitate for 1 min, and the floating liquid was blotted with filter paper again. After that, the sample was dried at room temperature for several minutes. Finally, transmission electron microscopy (TEM) imaging was performed under the conditions of 80 - 120 kV, and the observation was carried out under the transmission electron microscope. The results are shown in Figure 5 A, and it can be observed that an obvious PDA coating was formed on the surface of the drug-resistant Escherichia coli.

[0077] Example 5

[0078] In this example, the ability of Apt in the DNA hydrogel to capture drug-resistant bacteria was explored. The steps are as follows:

[0079] Add 10 μL of T2-Cy5 (200 mM) and 100 μL of GFP fluorescent bacteria (OD = 0.4) to 100 μL of DNA hydrogel, incubate at room temperature in the dark for 30 min, and then wash the DNA hydrogel three times with PBS. Among them, T2-Cy5 is a nucleic acid sequence that can specifically bind to the DNA hydrogel and is modified with a Cy5 red fluorescent dye. The GFP fluorescent bacteria are fluorescent bacteria that can express the green fluorescent protein GFP by themselves and can exhibit green fluorescence under excitation at an appropriate wavelength. Observe the washed DNA hydrogel under a laser scanning confocal microscope, and the results are as Figure 6 shown in A. The DNA hydrogel (red) has a significant ability to capture bacteria (green).

[0080] Example 6

[0081] In this example, the anti-drug-resistant bacteria performance of the DNA hydrogel was explored, and the steps were as follows:

[0082] Incubate 100 μL of DNA hydrogel, 9.2 μL of K + (3 M) and 8 μL of Hemin at 37 °C for 10 min to form a G4 / Hemin composite structure, then add 20 μL of drug-resistant Escherichia coli (105 CFU / mL), 10.8 μL of HEPES buffer solution (pH 6.5), 2 μL of H2O2 (100 mM), and 50 μL of DA (200 mM). After reacting at 37 °C for 30 min, irradiate with an 808 nm infrared laser at 1800 mA for 10 min, then take 50 μL of the treated sample and evenly inoculate it on Luria-Bertani (LB) medium, and culture it at 37 °C for 1 d and take pictures for recording. As Figure 7 shown in group ⑥, after the DNA hydrogel catalyzes the formation of a PDA coating layer on the surface of drug-resistant bacteria, under near-infrared light (NIR) irradiation, it exhibits significant antibacterial activity.

[0083] Comparative Example 1

[0084] Adopt the steps in Example 4, but do not pre-treat the drug-resistant Escherichia coli, which is used as the control group of Example 4. As Figure 5 shown in B, the surface of the drug-resistant Escherichia coli is smooth without a PDA coating layer.

[0085] Comparative Example 2

[0086] Adopt the steps in Example 5, but replace the DNA hydrogel with a DNA hydrogel without an Apt functional module - NApt (DNAhyd-NApt). The specific preparation method of the DNA hydrogel-NApt is basically the same as that in Example 1, and the difference is that DNA template strand 1 is replaced with DNA template strand 3. The results are as Figure 6As shown in B, the DNA hydrogel without Apt cannot specifically recognize and capture bacteria.

[0087] Comparative Example 3

[0088] The steps in Example 6 were adopted, but there were changes in the antibacterial treatment process, and the corresponding components of the material were deleted to be used as the control group of the final group ⑥. The specific groups were (①: bacteria, ②: bacteria + H2O2, ③: bacteria + DA, ④: bacteria + H2O2 + DNAhyd, ⑤: bacteria + H2O2 + DNAhyd - NApt, ⑥: bacteria

[0089] + H2O2 + DA + DNAhyd). The results are shown in Figure 7 , and it can be seen that the antibacterial effect of the final group ⑥ is the best.

[0090] In summary, the present invention forms a three-dimensional network structure through the rolling circle amplification technology, integrating the nucleic acid aptamer targeting module and the G-quadruplex catalytic module. At the site of drug-resistant bacterial infection, the nucleic acid aptamer can specifically bind to the peptidoglycan on the surface of bacteria, achieving precise capture of drug-resistant bacteria. The G4 sequence forms a composite structure with hemin with peroxidase-like activity under the induction of potassium ions. This composite structure uses the endogenous hydrogen peroxide in the infected microenvironment to catalyze the in-situ polymerization of dopamine on the surface of bacteria to form a polydopamine coating layer. The PDA coating layer generates reactive oxygen species under the excitation of near-infrared light and efficiently kills drug-resistant bacteria through the photothermal effect, realizing a three-level control mechanism of precise targeting, intelligent catalysis, and photothermal effect.

[0091] Based on the ideal embodiments of the present invention as an inspiration, through the above description, for those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

[0092] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a smart catalytic DNA hydrogel, characterized in that: The following steps are involved: S1. providing two circular DNA template chains, wherein circular DNA template chain 1 comprises a nucleic acid aptamer sequence that specifically targets a bacterial surface component, and circular DNA template chain 2 comprises a sequence that can form a G-quadruplex; S2, extending the circular DNA template chain by rolling circle amplification reaction to form a DNA hydrogel with a three-dimensional cross-linked network structure; S3, mixing the DNA hydrogel with potassium ions and hemin, so that the G-quadruplex sequence is combined with the hemin to form a composite catalytic unit with peroxidase-like activity.

2. The method for preparing a smart catalytic DNA hydrogel according to claim 1, characterized in that: In the step S1, the sequence of the DNA template chain 1 is shown as SEQ ID NO:1, and the sequence of the DNA template chain 2 is shown as SEQ ID NO:

2.

3. The method for preparing a smart catalytic DNA hydrogel according to claim 1, characterized in that: In step S1, the nucleic acid aptamer sequence specifically targets bacterial peptidoglycan, and the polymerase used in the rolling circle amplification reaction is a strand displacement DNA polymerase.

4. The smart catalytic DNA hydrogel and its preparation method and application according to claim 1, characterized in that: In step S1, the circular DNA template chain comprises the following steps: S11, phosphorylation of the linear DNA template chain; S12, annealing the phosphorylated linear DNA template with the complementary primer and forming a circular structure through ligase catalysis.

5. The smart catalytic DNA hydrogel and its preparation method and application according to claim 4, characterized in that: In the step S11, the phosphorylation treatment is performed in a reaction system containing ATP; in the step S12, the ligase is T4 DNA ligase.

6. The smart catalytic DNA hydrogel and its preparation method and application according to claim 1, characterized in that: In step S3, the concentration of potassium ions is 100-200 mM, and the concentration of hemin is 40-100 μM.

7. A smart catalytic DNA hydrogel, characterized in that: Prepared by the preparation method according to any one of claims 1 to 6, the DNA hydrogel comprises: Nucleic acid aptamer functional module, used to specifically capture target bacteria; The G-quadruplex / hemin composite catalytic unit can catalyze the in situ polymerization of dopamine in an acidic microenvironment to form a polydopamine coating; The microstructure of the DNA hydrogel is a network void structure.

8. The smart catalytic DNA hydrogel according to claim 7, characterized in that: The polydopamine coating layer produces a photothermal effect under near-infrared light irradiation.

9. Use of the smart catalytic DNA hydrogel according to any one of claims 7 to 8 in the preparation of a drug for treating drug-resistant bacterial infections.

10. The use according to claim 9, characterized in that: The drug works through the following mechanisms: Targeted capture of drug-resistant bacteria using nucleic acid aptamers; In the bacterial infection microenvironment, the composite catalytic unit catalyzes the polymerization of dopamine to form a polydopamine coating layer; The polydopamine coating layer is excited by near-infrared light to produce a photothermal effect, thereby killing drug-resistant bacteria.

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