Active oxygen tolerance programmable DNA origami nanoparticles and preparation method thereof

Abstract

The invention relates to active oxygen tolerance programmable DNA origami nanoparticles and a preparation method thereof.The preparation method comprises the following steps that in a buffer solution, an M13 support and a corresponding stapler chain are mixed according to the molar ratio of 1: 5, and then annealing is conducted; and after annealing, carrying out ultrafiltration treatment on the synthesized DNA origami nanoparticles to remove free stapler chains so as to obtain the DNA origami nanoparticles with programmable active oxygen tolerance. The invention further provides application of the active oxygen tolerance programmable DNA origami nanoparticles prepared on the basis of the method in preparation of detection sensors. Compared with the prior art, the relationship between the number of the cracks in the DNA origami and the tolerance to the active oxygen is found for the first time. Then, the found rule has important guiding significance for improving DNA origami and improving the active oxygen tolerance, and the biological application scene of the DNA nanostructure is greatly expanded.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a DNA origami nanoparticle with programmable active oxygen tolerance and a preparation method thereof. BACKGROUND

[0002] Active oxygen widely refers to free radicals and non-free radicals derived from oxygen, including superoxide anion (O 2- ), hydrogen peroxide (H2O2), hydroxyl radical (OH - ), ozone (O3) and singlet oxygen ( 1 O2), which have high chemical reactivity due to containing unpaired electrons. Active oxygen plays an important role in a series of important redox-dependent signal pathways and is crucial for cell and organism growth and survival.

[0003] DNA, as a basic genetic material, is used to store genetic information. DNA material has the characteristics of high precision and high controllability of Watson-Crick base pairs, and has the potential for nanometerization. In the 1980s, Seeman et al. first proposed the synthesis rules of DNA nanostructure, which triggered the vigorous development of DNA nanotechnology. In the following decades, DNA nanostructure technology has been rapidly developed and improved, and various DNA nanostructures have been designed into common polymer materials and widely used, forming complex nanoparticles with specific purposes in the field of chemical and biological materials. With the promotion of DNA nanotechnology, DNA as a genetic material has been applied to different scientific fields such as tissue regeneration, disease prevention, inflammation inhibition, biological imaging, biological sensing, diagnosis, anti-tumor drug delivery and treatment.

[0004] DNA nanostructure inherits the reducibility of DNA material and can react with active oxygen species to cause damage and collapse of the DNA nanostructure. Improving the tolerance of DNA nanostructure to active oxygen is crucial for the application of DNA nanostructure. SUMMARY

[0005] There is a lack of DNA origami nanoparticles with high active oxygen tolerance in the prior art, and the purpose of the present application is to provide a DNA origami nanoparticle with programmable active oxygen tolerance and a preparation method thereof.

[0006] By programming the number of cracks of the DNA origami nanoparticle, the present application can realize the regulation of the active oxygen tolerance of the DNA origami nanoparticle, thereby enhancing the stability of the DNA origami nanoparticle in a biological background.

[0007] Based on this, the application prepares a series of DNA origami nanoparticles with different numbers of cracks, and finds that the number of cracks of the DNA origami nanoparticle is inversely related to the active oxygen tolerance. This design rule has important guiding significance for designing a DNA nanostructure with high active oxygen tolerance.

[0008] The object of the application can be achieved by the following technical solutions:

[0009] The application first provides a preparation method of a DNA origami nanoparticle with programmable active oxygen tolerance, comprising the following steps:

[0010] In a buffer, M13 scaffolds (20 nM) are mixed with corresponding stapler chains at a molar ratio of 1:5, and then annealed;

[0011] After annealing, the synthesized DNA origami nanoparticle is subjected to ultrafiltration treatment to remove free stapler chains, so as to obtain a DNA origami nanoparticle with programmable active oxygen tolerance.

[0012] In an embodiment of the application, the M13 scaffold is single-stranded, circular viral DNA isolated from M13mp18, containing 7249 bases (p7249), and is a M13 lac bacteriophage vector.

[0013] In an embodiment of the application, the buffer is 1xTAE / Mg 2+ Buffer, the component group is: 40mM Tris, 2mM EDTA, 12.5mM magnesium acetate, pH 8.0.

[0014] In an embodiment of the application, the operation condition of annealing is as follows: the annealing process is carried out in a PCR thermal cycler (Bio-Rad, T100) according to the following scheme: 20 minutes at 65℃, then from 60℃ to 40℃ at a rate of 20 minutes / ℃, and then from 40℃ to 25℃ at a rate of 15 minutes / ℃.

[0015] In an embodiment of the application, the ultrafiltration treatment is carried out under the following conditions: 3-4 times of ultrafiltration at 3500g for 5 minutes in a 100kDa MWCO ultrafiltration tube (Amicon® Ultra 0.5mL centrifugal filter). Ultra 0.5mL centrifugal filter).

[0016] In an embodiment of the application, the DNA origami nanoparticle with programmable active oxygen tolerance is a DNA origami nanoparticle containing 155 cracks, expressed as DNA origami nanoparticle-155.

[0017] The information of the staple strand sequence of the DNA origami nanoparticle-155 is generated according to the Cadnano2 software, and a total of 183 staple strands are mixed in an equimolar ratio, and the total staple strands are mixed with the M13 scaffold in a molar ratio of 1:5, and after annealing, the DNA origami nanoparticle-155 is obtained.

[0018] Further, the DNA origami nanoparticle-155 is designed and generated by using the open source software Cadnano2, and the specific process is as follows:

[0019] Open the software Cadnano2, select the square lattice, select 30 circles from left to right, and increase the length of each helix to 383 bases;

[0020] After filling the M13 chain from the beginning to the end of each row, the connection between the helices in the middle and both ends is carried out, so that the first position is connected to form a complete chain;

[0021] Then draw the staple strand, in the main part, each staple strand penetrates four rows, that is, contains three cross points, the first row and the fourth row have 8 bases respectively, and the middle two rows have 16 bases respectively, so that each staple strand is connected at the head and tail, and covers the entire M13 structure, and the design of the origami structure is completed;

[0022] Use the sequence of M13 to fill in to generate the sequence of each staple strand.

[0023] According to the CaDNAno design diagram, Figure 2 ), the structure contains 155 nick numbers. In an embodiment of the present application, the active oxygen tolerance programmable DNA origami nanoparticle is a DNA origami nanoparticle containing 191 cuts, expressed as DNA origami nanoparticle-191.

[0024] The DNA origami nanoparticle-191 adopts the reported classic triangular DNA origami structure (Jiang, D., Ge, Z., Im, HJ. et al. DNA origami nanostructures can exhibit preferential renal uptake and alleviate acute kidney injury. Nat Biomed Eng 2, 865-877 (2018)). According to the triangular DNA origami structure, the sequence of all the staple strands of the DNA origami nanoparticle-191 is generated in the Cadnano2 software, a total of 208 staple strands are mixed in an equimolar ratio, and all the staple strands are mixed with the M13 scaffold in a molar ratio of 1:5, and after annealing, the DNA origami nanoparticle-191 is obtained.

[0025] The CaDNAno design diagram of DNA origami nanoparticle-191 is as shown in Figure 1. Figure 3 The structure contains 191 nick numbers.

[0026] In an embodiment of the present application, the active oxygen tolerance programmable DNA origami nanoparticle is a DNA origami nanoparticle containing 290 cuts, expressed as DNA origami nanoparticle-290.

[0027] The sequence information of the stapler chain of DNA origami nanoparticle-290 is generated according to the Cadnano2 software, and there are 304 stapler chains. All the stapler chains are mixed in an equimolar ratio, and the total stapler chain is mixed with the M13 support in a molar ratio of 1:5. After annealing, DNA origami nanoparticle-290 is obtained.

[0028] The method for obtaining the sequence information of the stapler chain of DNA origami nanoparticle-290 is as follows:

[0029] Open the software Cadnano2, and on the basis of the structure of DNA origami nanoparticle-191, ensure the total number of cross points, and cut the stapler chains numbered 46, 42, 38, 09, 13, 17, 47, 39, 10, 18, 55, 48, 40, 11, 19, 25, 63, 61, 59, 56, 53, 49, 45, 41, 08, 16, 23, 28, 12, 20, 26, 30 into two sequences of 24 bases and 16 bases, to obtain all the stapler chains, a total of 304.

[0030] According to the CaDNAno design diagram of DNA origami nanoparticle-290, it can be known that Figure 4 The structure contains 290 nick numbers.

[0031] After the DNA origami nanoparticle is prepared in the present application, the DNA origami nanoparticle is also simulated and characterized. The specific method is as follows:

[0032] Thermodynamic fluctuations of DNA origami nanoparticles were investigated using the oxDNA simulation framework. The oxDNA framework employs a top-down parameterization approach, where each nucleotide represents a site with six anisotropic interactions, including excluded volume effects, stacking and cross-stacking interactions, hydrogen bonds, backbone connections, and electrostatic repulsion. The DNA origami nanoparticles were transformed into their corresponding structures in oxView, serving as the starting point for molecular simulations. These simulations underwent a two-step relaxation process. First, Monte Carlo (MC) simulations of structural stress were performed using the DNA_relax force field. Subsequently, additional relaxation was implemented in molecular dynamics simulations using the max_backbone_force option within the DNA2 force field. Molecular simulation conditions were set at 300 K, with a step size of 6.06 fs, and 1 × 10⁻⁶ simulations per system. 9 MD step size. To accelerate diffusion kinetics and improve sampling efficiency, the translational diffusion coefficient of the particles was set to 2.5. A Newtonian step of 10³ was used, with each step being 1 × 10⁻³. 4 Each step records one configuration. All simulations were performed in a monovalent salt environment of 1M NaCl. In molecular dynamics simulations, every 2 × 10⁻⁶ steps... 4 The potential energy of different structures is recorded in an energy file. Once equilibrium is reached, the simulated trajectory is analyzed using the oxDNA analysis script to calculate the root mean square fluctuation (RMSF) value and determine the centroid structure. The trajectory data is then extracted using oxView to produce high-quality visualizations.

[0033] After preparing the DNA origami nanoparticles in this invention, the tolerance of the DNA origami nanoparticles to reactive oxygen species was verified using atomic force microscopy. The specific method is as follows:

[0034] Prepare 1 mL of 1 mM CuCl2 solution and 1 mL of 0.5 M H2O2 solution. Then, add 30 μL of 0.5 M H2O2 and 30 μL of 20 nM DNA origami nanoparticle solution (i.e., a solution in which M13 scaffold and corresponding stapler strand are mixed in a buffer solution at a molar ratio of 1:5) to 210 μL of 1×TAE / Mg 2+ Mix the mixture on a stirrer, then gradually add 30 μL of 1 mM CuCl2 solution. AFM images were acquired at 20, 60, and 100 minutes. The average folded area at different reaction time points was quantified using ImageJ and used as the reactant residual concentration (C0) at that time point. t Using the area of ​​intact DNA origami nanoparticles as the initial reaction concentration (C0), and C... t / C0 represents the degree of degradation. Curve fitting of the results revealed that the degradation kinetics of DNA origami nanoparticles induced by reactive oxygen species follows a first-order kinetic reaction, i.e., ln(C0 / 20) = 0.t The slope of the equation is the kinetic constant k.

[0035] The application further provides the active oxygen tolerance programmable DNA origami nanoparticle prepared based on the method.

[0036] The application further provides application of the active oxygen tolerance programmable DNA origami nanoparticle prepared based on the method in preparation of a detection sensor.

[0037] In an embodiment of the application, the sensor is a DNA origami sensor for diagnosing early acute liver injury.

[0038] Compared with the prior art, the application has the following advantages and beneficial effects:

[0039] The application first discovers the relationship between the number of cracks in the DNA origami and the active oxygen tolerance thereof. Then, the discovered rule has important guiding significance for improving the active oxygen tolerance of the DNA origami, and greatly expands the biological application scenarios of the DNA nanostructure. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 FIG. 1 is a schematic diagram of a DNA origami sensor for diagnosing early acute liver injury. (Right) Synthesis of DNA origami sensors with different numbers of cracks. (Left) Mechanism diagram of the DNA origami sensor for diagnosing early acute liver injury.

[0041] Figure 2 FIG. 2 is a design of DNA origami nanoparticle-155. (A) Layout of the DNA origami nanoparticle-155 in the caDNAno software. (B) Design sketch of the DNA origami nanoparticle-155; the blue square represents a cut. (C) Structure sketch of the DNA origami nanoparticle-155 in the oxView software.

[0042] Figure 3 FIG. 3 is a design of DNA origami nanoparticle-191. (A) Layout of the DNA origami nanoparticle-191 in the caDNAno software. (B) Design sketch of the DNA origami nanoparticle-191; the blue square represents a cut. (C) Structure sketch of the DNA origami nanoparticle-191 in the oxView software.

[0043] Figure 4 FIG. 4 is a design of DNA origami nanoparticle-290. (A) Layout of the DNA origami nanoparticle-290 in the caDNAno software. (B) Design sketch of the DNA origami nanoparticle-290; the blue square represents a cut. (C) Structure sketch of the DNA origami nanoparticle-290 in the oxView software.

[0044] Figure 5 Synthesis and characterization of DNA origami nanoparticles with different number of cuts. (A) Schematic diagram of structure information of DNA origami nanoparticles with different number of cuts. (B) Root mean square fluctuation (RMSF) of DNA origami nanoparticles with different number of cuts. (C) Single base energy distribution of DNA origami nanoparticles with different number of cuts. (D) AFM images of DNA origami nanoparticles with different number of cuts.

[0045] Figure 6 Degradation sensitivity of DNA origami nanoparticles with different number of cuts to reactive oxygen species. (A) Schematic diagram of AFM imaging detection of DNA origami nanoparticles with different number of cuts degradation process induced by reactive oxygen species. (B) Upper panel: AFM images of DNA origami nanoparticle-155 reacting with reactive oxygen species for different time; Lower panel: Average area statistics of DNA origami nanoparticle-155 reacting with reactive oxygen species for different time in AFM images. (C) Upper panel: AFM images of DNA origami nanoparticle-191 reacting with reactive oxygen species for different time; Lower panel: Average area statistics of DNA origami nanoparticle-191 reacting with reactive oxygen species for different time in AFM images. (D) Upper panel: AFM images of DNA origami nanoparticle-290 reacting with reactive oxygen species for different time; Lower panel: Average area statistics of DNA origami nanoparticle-290 reacting with reactive oxygen species for different time in AFM images. (E) Reaction kinetics of DNA origami nanoparticles with reactive oxygen species. (F) First-order reaction kinetics of DNA origami nanoparticles with reactive oxygen species. (G) Apparent rate constant and half-life of DNA origami nanoparticles with reactive oxygen species. (H) Statistical percentage of <~6 nm degradation fragments of DNA origami nanoparticles reacting with reactive oxygen species for 100 min in AFM images. Data are mean ± standard deviation (n = 3), **p < 0.01. DETAILED DESCRIPTION

[0046] The present application will be further described in conjunction with the following examples and accompanying drawings, but the embodiments of the present application are not limited thereto. The drugs and reagents used in the examples can be purchased from chemical companies or can be obtained by preparing methods disclosed in the art.

[0047] In the following examples, the M13 scaffold is a single-stranded, circular viral DNA isolated from M13mp18, which contains a total of 7249 bases (p7249), and is a M13 lac bacteriophage vector. The purchase source is Baobio, product number 3518, M13mp18 Single Strand DNA (Virion DNA).

[0048] Example 1

[0049] A schematic diagram of a DNA origami sensor for diagnosing early acute liver injury is shown in Figure 1 .

[0050] Design of DNA origami nanoparticles.

[0051] In this embodiment, triangular DNA origami was selected as a fixed topological shape to study the relationship between the number of nicks and reactive oxygen species tolerance. Using CaDNAno2, the number of nicks in the DNA origami nanoparticles can be controlled by changing the staple chain length. Considering the influence of the staple chain length range (18-49 nt) and the significant differences in DNA origami nanoparticles, this embodiment designed three types of triangular DNA origami nanoparticles with different numbers of nicks: DNA origami nanoparticle-155 (containing 155 nicks), DNA origami nanoparticle-191 (containing 191 nicks), and DNA origami nanoparticle-290 (containing 290 nicks).

[0052] The stapler strand sequence information of DNA Origami Nanoparticle-155 was generated by Cadnano2 software, with a total of 183 stapler strands. All stapler strands were mixed in an equimolar ratio, and the total stapler strands were mixed with the M13 scaffold in a molar ratio of 1:5. After annealing, DNA Origami Nanoparticle-155 was obtained.

[0053] DNA Origami Nanoparticles-155 were designed and generated using the open-source software Cadnano2. The specific process is as follows: Open Cadnano2, select the square lattice, select 30 circles from left to right, and increase the length of each helix to 383 bases; after filling each row with M13 chains from beginning to end, connect the helices in the middle and at both ends to form a complete chain; then draw the stapler chains. In the main body, each stapler chain runs through four rows, containing three intersections. The first and fourth rows each have 8 bases, and the two middle rows each have 16 bases. In this way, each stapler chain is connected end to end, covering the entire M13 structure to complete the origami structure design; use the M13 sequence to fill and generate the sequence of each stapler chain. According to its CaDNAno design diagram, ( Figure 2 This structure contains 155 nicks.

[0054] The stapler strand sequence information of DNA origami nanoparticle-155 is shown in SEQ ID NO.1 to SEQ ID NO.183 in the sequence listing, totaling 183 short stapler strands.

[0055] DNA origami nanoparticles-191 utilize the previously reported classic triangular DNA origami structure (Jiang, D., Ge, Z., Im, HJ. et al. DNA origaminanostructures can exhibit preferential renal uptake and alleviate acute kidney injury. Nat Biomed Eng 2, 865–877 (2018).). Based on its triangular DNA origami structure, all stapler strand sequences of DNA origami nanoparticles-191 were generated in Cadnano2 software, totaling 208 stapler strands. Different stapler strands were mixed in an equimolar ratio, and all stapler strands were mixed with the M13 scaffold at a molar ratio of 1:5. After annealing, DNA origami nanoparticles-191 were obtained. The CaDNAno design diagram of DNA origami nanoparticles-191 is shown below. Figure 3 The structure contains 191 nicks.

[0056] The stapler strand sequence information of DNA Origami Nanoparticle-290 was generated using Cadnano2 software, with a total of 304 stapler strands. All stapler strands were mixed in an equimolar ratio, and the total stapler strands were mixed with the M13 scaffold at a molar ratio of 1:5. After annealing, DNA Origami Nanoparticle-290 was obtained.

[0057] The method for obtaining the stapler strand sequence information of DNA Origami Nanoparticle-290 is as follows: Open the software Cadnano2. Based on the structure of DNA Origami Nanoparticle-191, while ensuring the total number of crossovers remains unchanged, the stapler strands numbered 46, 42, 38, 09, 13, 17, 47, 39, 10, 18, 55, 48, 40, 11, 19, 25, 63, 61, 59, 56, 53, 49, 45, 41, 08, 16, 23, 28, 12, 20, 26, and 30 are divided into two segments of 24 bases and 16 bases, obtaining all stapler strands, totaling 304. According to the CaDNAno design diagram of DNA Origami Nanoparticle-290, (… Figure 4 This structure contains 290 nicks.

[0058] The stapler strand sequence information of DNA origami nanoparticle-155 is shown in Table 1.

[0059] Table 1. Staple strand sequence information of DNA origami nanoparticles-155

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] The stapler strand sequence information of DNA origami nanoparticle-191 is shown in Table 2.

[0067] Table 2 Sequence information of DNA origami nanoparticles-191

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] The stapler strand sequence information of DNA origami nanoparticle-290 is shown in Table 3.

[0075] Table 3 Sequence information of DNA origami nanoparticles-290

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Example 2

[0085] Synthesis of DNA origami nanoparticles.

[0086] At 1×TAE / Mg 2+In a buffer solution (40 mM Tris, 2 mM EDTA, 12.5 mM magnesium acetate, pH 8.0), the M13 scaffold (20 nM) was mixed with the corresponding stapler strand at a total molar ratio of 1:5. The annealing process was performed in a PCR thermal cycler (Bio-Rad, T100) according to the following protocol: 65 °C for 20 min, then cooled from 60 °C to 40 °C at a rate of 20 min / °C, and then cooled from 40 °C to 25 °C at a rate of 15 min / °C. After annealing, the synthesized DNA origami nanoparticles were filtered through 100 kDa MWCO ultrafiltration tubes (…). Ultrafiltration (3500g for 3-4 times, 5 minutes each time) in an Ultra 0.5mL centrifugal filter to remove free stapler chains.

[0087] Example 3

[0088] Simulated characterization of DNA origami nanoparticles.

[0089] Thermodynamic fluctuations of DNA origami nanoparticles were investigated using the oxDNA simulation framework. The oxDNA framework employs a top-down parameterization approach, where each nucleotide represents a site with six anisotropic interactions, including excluded volume effects, stacking and cross-stacking interactions, hydrogen bonds, backbone connections, and electrostatic repulsion. The DNA origami nanoparticles were transformed into their corresponding structures in oxView, serving as the starting point for molecular simulations. These simulations underwent a two-step relaxation process. First, Monte Carlo (MC) simulations of structural stress were performed using the DNA_relax force field. Subsequently, additional relaxation was implemented in molecular dynamics simulations using the max_backbone_force option within the DNA2 force field. Molecular simulation conditions were set at 300 K, with a step size of 6.06 fs, and 1 × 10⁻⁶ simulations per system. 9 MD step size. To accelerate diffusion kinetics and improve sampling efficiency, the translational diffusion coefficient of the particles was set to 2.5. A Newtonian step of 10³ was used, with each step being 1 × 10⁻³. 4 Each step records one configuration. All simulations were performed in a monovalent salt environment of 1M NaCl. In molecular dynamics simulations, every 2 × 10⁻⁶ steps... 4 The potential energy of different structures is recorded in an energy file. Once equilibrium is reached, the simulated trajectory is analyzed using the oxDNA analysis script to calculate the root mean square fluctuation (RMSF) value and determine the centroid structure. The trajectory data is then extracted using oxView to produce high-quality visualizations.

[0090] First, coarse-grained models of these three DNA origami nanoparticles were constructed using oxDNA software to study their molecular motion and stability. Figures 2-4As shown, the simulation models of these three structures all exhibit a two-dimensional triangular topological structure, which theoretically demonstrates that reasonable changes in the number and location of the cracks do not significantly affect DNA assembly behavior.

[0091] Then, the volatility of these three structures is analyzed using root mean square volatility (RMSF). Figure 5 As shown in Figure AB, the average RMSF values ​​of DNA origami nanoparticles-155,-191, and-290 are 3.419, 3.277, and 3.284, respectively. These values ​​demonstrate relatively consistent volatility among the three structures, indicating similar atomic vibration amplitudes during simulation. Furthermore, the average RMSF value of DNA origami nanoparticle-290 is slightly higher than the other two structures, suggesting slightly stronger overall volatility. This slight difference may be due to the increased number of staple chains in the structure.

[0092] To further confirm the stability of these three origami structures, the single-base energy distribution of these three origami nanoparticles after relaxation was studied using oxDNA. Figure 5 As shown in Figure C, the relaxed single-base energies of DNA origami nanoparticles-155,-191, and-290 are -15.23 K. b T, -15.22K b T, -15.19K b Among the T values, DNA origami nanoparticle-290 has the highest energy value, indicating that it has a higher energy state than DNA origami nanoparticle-155 and DNA origami nanoparticle-191. In summary, theoretical simulations preliminarily confirm that changes in the number and location of the gaps do not affect the assembly behavior of DNA origami.

[0093] Example 4

[0094] Atomic force microscopy was used to verify the tolerance of DNA origami nanoparticles to reactive oxygen species.

[0095] Prepare 1 mL of 1 mM CuCl2 solution and 1 mL of 0.5 M H2O2 solution. Then, add 30 μL of 0.5 M H2O2 and 30 μL of 20 nM DNA origami nanoparticle solution to 210 μL of 1×TAE / Mg solution. 2+ Mix the mixture on a stirrer, then gradually add 30 μL of 1 mM CuCl2 solution. AFM images were acquired at 20, 60, and 100 minutes. The average folded area at different reaction time points was quantified using ImageJ and used as the reactant residual concentration (C0) at that time point. t Using the area of ​​intact DNA origami nanoparticles as the initial reaction concentration (C0), and C...t / C0 represents the degree of degradation. Curve fitting of the results revealed that the degradation kinetics of DNA origami nanoparticles induced by reactive oxygen species follows a first-order kinetic reaction, i.e., ln(C0 / 20) = 0. t The reaction time ( / C0) is linearly related to the reaction time. The slope of the equation is the kinetic energy constant k.

[0096] This study elucidates the relationship between the number of rifts in DNA origami and reactive oxygen species (ROS) tolerance by investigating the degradation kinetics of ROS on DNA origami nanoparticles with varying numbers of rifts. Copper-mediated Fenton reactions were employed to generate ROS. Figure 6 A). Mica sheets with adsorbed DNA origami nanoparticles were placed in a buffer solution containing hydrogen peroxide (H2O2), followed by the slow addition of copper salt and reaction under magnetic stirring. DNA origami nanoparticle samples at different reaction time points were selected, and the topological structure of the DNA origami nanoparticles was visualized using AFM to illustrate the reaction behavior of DNA origami nanoparticles with reactive oxygen species.

[0097] like Figure 6 As shown in Figure BD, under the presence of reactive oxygen species (ROS), the topological area of ​​all three DNA origami nanoparticles decreased to varying degrees, indicating morphological disruption. Quantitative analysis of the average area of ​​the DNA origami nanoparticles at different time points using ImageJ software revealed that the degradation kinetics of ROS on all three types of DNA origami nanoparticles exhibited first-order reaction kinetics. Specifically, the degradation rates of DNA origami nanoparticle-155, DNA origami nanoparticle-191, and DNA origami nanoparticle-290 were 1.1 × 10⁻⁶, respectively. -3 min -1 5.4*10 -3 min -1 23.5*10 -3 min -1 This indicates that as the number of rifts increases, DNA origami nanoparticles exhibit higher ROS degradation sensitivity. Notably, after reacting with ROS for 100 min, DO-290 was almost completely degraded, with a topological area reduction of approximately ~99.9%, which is 7.88 times that of DO-155 and 2.79 times that of DO-191.

[0098] The study focused on extracting fragment information smaller than 6 nm during the degradation process of DNA origami nanoparticles. Statistical results showed that after 100 minutes of reaction with reactive oxygen species (ROS), the proportion of particles smaller than 6 nm in the DNA origami nanoparticle-290 sample significantly increased to 72.4%, which was significantly higher than the 49.6% in the DNA origami nanoparticle-191 sample and the 35.9% in the DNA origami nanoparticle-155 sample. This difference indicates that the degradation rate of the DNA origami nanoparticle-290 sample was faster than other samples, which is related to its unique structural characteristics. In summary, by controlling the number of cleavage points in the DNA origami nanoparticles, the degradation tolerance of DNA origami nanoparticles to ROS can be modulated.

Claims

1. A method for preparing DNA origami nanoparticles with programmable reactive oxygen species tolerance, characterized in that, Includes the following steps: In a buffer solution, the M13 scaffold is mixed with the corresponding stapler chain at a molar ratio of 1:5, and then annealed. After annealing, the synthesized DNA origami nanoparticles were subjected to ultrafiltration to remove free stapler chains, resulting in DNA origami nanoparticles with programmable reactive oxygen species tolerance.

2. The method for preparing DNA origami nanoparticles with programmable reactive oxygen species tolerance according to claim 1, characterized in that, The buffer solution is 1×TAE / Mg. 2+ The buffer solution consists of 40 mM Tris, 2 mM EDTA, 12.5 mM magnesium acetate, and has a pH of 8.

0.

3. The method for preparing DNA origami nanoparticles with programmable reactive oxygen species tolerance according to claim 1, characterized in that, The annealing conditions are as follows: 20 minutes at 65°C, then decreasing from 60°C to 40°C at a rate of 20 minutes / °C, and then decreasing from 40°C to 25°C at a rate of 15 minutes / °C.

4. The method for preparing DNA origami nanoparticles with programmable reactive oxygen species tolerance according to claim 1, characterized in that, The conditions for ultrafiltration are as follows: 3500g ultrafiltration is performed 3-4 times in a 100kDa MWCO ultrafiltration tube for 5 minutes each time.

5. The method for preparing DNA origami nanoparticles with programmable reactive oxygen species tolerance according to claim 1, characterized in that, The reactive oxygen species tolerance programmable DNA origami nanoparticles are DNA origami nanoparticles containing 155 slits, expressed as DNA origami nanoparticle-155. The stapler strand sequence information of DNA origami nanoparticle-155 is generated according to Cadnano2 software, with a total of 183 stapler strands. All stapler strands are mixed in an equimolar ratio, and the total stapler strands are mixed with the M13 scaffold at a molar ratio of 1:

5. After annealing, DNA origami nanoparticle-155 is obtained.

6. The method for preparing DNA origami nanoparticles with programmable reactive oxygen species tolerance according to claim 1, characterized in that, The reactive oxygen species tolerance programmable DNA origami nanoparticles are DNA origami nanoparticles containing 191 slits, expressed as DNA origami nanoparticle-191. DNA origami nanoparticle-191 adopts a classic triangular DNA origami structure. Based on its triangular DNA origami structure, all the stapler strand sequences of DNA origami nanoparticle-191 are generated in Cadnano2 software, totaling 208 stapler strands. Different stapler strands are mixed in an equimolar ratio. All stapler strands are mixed with the M13 scaffold in a molar ratio of 1:

5. After annealing, DNA origami nanoparticle-191 is obtained.

7. The method for preparing DNA origami nanoparticles with programmable reactive oxygen species tolerance according to claim 1, characterized in that, The reactive oxygen species tolerance programmable DNA origami nanoparticles are DNA origami nanoparticles containing 290 slits, expressed as DNA origami nanoparticle-290. The stapler strand sequence information of DNA origami nanoparticle-290 is generated according to Cadnano2 software, with a total of 304 stapler strands. All stapler strands are mixed in an equimolar ratio, and the total stapler strands are mixed with the M13 scaffold at a molar ratio of 1:

5. After annealing, DNA origami nanoparticle-290 is obtained.

8. DNA origami nanoparticles with programmable reactive oxygen species tolerance prepared by the method according to any one of claims 1-7.

9. The application of the reactive oxygen species tolerance programmable DNA origami nanoparticles as described in claim 9 in the preparation of detection sensors.

10. The application according to claim 9, characterized in that, The sensor is a DNA origami sensor used to diagnose early acute liver injury.

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