A DNA origami structure based on functionalized magnetic nanoparticles, preparation method and application

Abstract

The present invention relates to the field of DNA nanotechnology, and discloses a DNA origami structure based on functionalized magnetic nanoparticles, a preparation method and an application thereof, comprising the following steps: step 1: constructing a DNA origami structure with a cavity by a self-assembly method; the short chains in the DNA origami structure include a first short chain, a second short chain and a third short chain; step 2: connecting a first sequence functionalized with a thiol group to the first short chain, and chelating ferrous ions on the first sequence to obtain a thiol-modified DNA origami structure; step 3: reducing the chelated ferrous ions on the thiol-modified DNA origami structure to obtain a magnetic functionalized DNA origami structure loaded with Fe2O3 particles internally; step 4: connecting an antibody to the second short chain in the DNA origami structure through base complementary pairing to obtain a desired DNA origami structure; the present invention combines DNA origami with magnetic nanoparticles, and obtains efficient and controllable biomagnetic beads through dual fixation and antibody modification, breaking through the traditional application scenarios of DNA origami.

Description

Technical Field

[0001] The present invention relates to the field of DNA nanotechnology, and in particular to a DNA origami structure based on functionalized magnetic nanoparticles, a preparation method and an application thereof. Background Art

[0002] Currently, early disease detection relies primarily on the detection of specific biomarkers, such as S100-β, a protein associated with brain damage, thrombomodulin, and D-dimer, which are associated with thrombosis. These biomarkers are often detected using magnetic nanoparticle adsorption methods based on magnetic beads. Magnetic beads are a special class of nanomaterials, typically ranging in diameter from a few hundred nanometers to a few microns, and possess superparamagnetic properties. Under the influence of an external magnetic field, the beads rapidly align in the direction of the field and quickly return to a dispersed state upon removal. When antibodies are loaded onto their surfaces, they rapidly bind to specific antigens. Magnetic separation techniques are then used to separate the beads from the substance being tested. By labeling antibodies or antigens with readily detectable substances and detecting changes in these markers, the binding reaction between the antigen and antibody can be indirectly reflected, enabling the precise detection of trace amounts of antigen or antibody. This method offers significant advantages in terms of improved sensitivity, specificity, and ease of use, and is widely used for early disease diagnosis. However, China still relies on importing uniformly sized magnetic bead carriers, and the antigens loaded on the bead surface are often difficult to quantify. This often requires repeated trials and excessive incubations to obtain beads with the appropriate antigen loading. During this process, batch-to-batch variations in carriers can significantly affect the accuracy of test results.

[0003] DNA origami technology is a DNA-based bio-nanotechnology that uses a circular single-stranded DNA and hundreds of precisely designed short-chain DNAs to self-assemble into predetermined nanostructures through programmed annealing under specific conditions. The programmability of this technology enables the DNA origami structure to be folded into a specific shape strictly in accordance with the design rules, thereby ensuring its excellent uniformity, and the particle size can be precisely controlled at the nanometer level. In addition, each short chain of the DNA origami structure has a fixed position in the design. This nanoscale addressability allows guest molecules to be accurately loaded to the specified position, achieving high-precision spatial positioning. The number of short chain sequences in each DNA origami structure is fixed, and the occupancy rate of base complementary pairing exceeds 90%, ensuring that the number of guest molecules loaded through short chain complementarity is uniform and has quantitative loading capacity.

[0004] The application of magnetic beads in immunodiagnosis faces multiple challenges: First, the stability of the functionalization of the magnetic bead surface is poor, which may cause the modification to fall off or degrade, thereby affecting the binding efficiency of the antigen or antibody; second, nonspecific binding may lead to an increase in background signal, reducing the sensitivity and accuracy of detection; third, the stability of the antigen or antibody is easily affected by environmental factors, which may lead to a decrease in its activity; too high or too low loading density of antigen or antibody on the magnetic bead surface may affect the efficiency of the binding reaction, and thus affect the accuracy of the results. Differences in binding specificity and affinity may also affect the sensitivity of the detection; and differences between batches of magnetic beads often cause inconsistent performance, affecting the repeatability of the experiment. In addition, the aggregation of magnetic beads may also lead to reduced reaction efficiency and affect the diagnostic effect.

[0005] The present invention innovatively uses DNA origami structures to replace magnetic beads, overcoming the limitations of traditional magnetic beads. Summary of the Invention

[0006] In response to the technical problems existing in the prior art, the present invention provides a DNA origami structure based on functionalized magnetic nanoparticles, a preparation method and an application thereof.

[0007] The technical solution adopted by the present invention is: a method for preparing a DNA origami structure based on functionalized magnetic nanoparticles, comprising the following steps:

[0008] Step 1: Construct a DNA origami structure with a cavity by self-assembly. The DNA origami structure is formed by a scaffold chain and short chains that are complementary to the scaffold chain. The short chains include a first short chain extending into the cavity, a second short chain extending out of the cavity, and a third short chain.

[0009] Step 2: Connecting the thiol-functionalized first sequence to the first short chain through base complementary pairing, and chelating ferrous ions on the first sequence to obtain a thiol-modified DNA origami structure;

[0010] Step 3: reducing the ferrous ions chelated on the thiol-modified DNA origami structure obtained in step 2 to obtain a magnetic functionalized DNA origami structure loaded with Fe2O3 particles;

[0011] Step 4: Connect the antibody to the second short chain of the DNA origami structure obtained in step 3 through base complementary pairing to obtain the desired DNA origami structure.

[0012] Furthermore, the DNA origami structure in step 1 has a three-dimensional tubular structure.

[0013] Furthermore, the self-assembly conditions in step 1 are as follows:

[0014] Keep warm at 65°C for 15 min;

[0015] The temperature was lowered to 50 °C and then to 40 °C at a rate of 0.1 °C per 39.6 min;

[0016] The temperature was lowered to 15°C at a rate of 1°C per 2.4 minutes.

[0017] Furthermore, the specific process in step 2 is as follows:

[0018] Restore the first sequence;

[0019] Mixing the DNA origami structure obtained in step 1 with the reduced first sequence, and allowing the reaction to fully proceed to obtain a thiol-modified DNA origami structure;

[0020] The thiol-modified DNA origami structure is fully mixed with a ferrous ion solution to react, thereby obtaining a DNA origami structure chelating ferrous ions.

[0021] Furthermore, the restoration process in step 3 is as follows:

[0022] The thiol-modified DNA origami structure obtained in step 2 is placed in a NaBH4 solution, and after sufficient reaction, a magnetic functionalized DNA origami structure can be obtained.

[0023] Furthermore, in step 4, the antibody is connected by a biotin-streptavidin coupling method.

[0024] A DNA origami structure based on functionalized magnetic nanoparticles has a three-dimensional tubular structure, is internally coupled with magnetic nanoparticles, and is externally loaded with antibodies.

[0025] An application of a DNA origami structure based on functionalized magnetic nanoparticles, wherein the DNA origami structure is used to prepare biological magnetic beads.

[0026] Furthermore, the biomagnetic beads are used in a protein detection kit.

[0027] Furthermore, the antibody in the DNA origami structure is a specific antibody for the antigen to be detected.

[0028] The beneficial effects of the present invention are:

[0029] (1) The present invention combines DNA origami with magnetic nanoparticles, and through dual fixation and antibody modification, obtains efficient and controllable biomagnetic beads, breaking through the traditional application scenarios of DNA origami;

[0030] (2) The present invention precisely fixes magnetic nanoparticles on the DNA origami structure through chain complementarity and metal chelation;

[0031] (3) The present invention integrates the specific antibody of the antigen to be detected into the DNA origami structure through the biotin-streptavidin interaction to obtain precisely controllable biological magnetic beads. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of gel electrophoresis characterization of the DNA origami structure obtained in step 1 of Example 1 of the present invention.

[0033] Figure 2 The chelated Fe in step 3 of the embodiment of the present invention 2+ Schematic diagram of gel electrophoresis characterization of DNA origami structures.

[0034] Figure 3 Schematic diagram of gel electrophoresis characterization of the magnetically functionalized DNA origami structure obtained in step 3 of the embodiment of the present invention.

[0035] Figure 4 Schematic diagram of transmission electron microscopy characterization of the DNA origami structure of functionalized magnetic nanoparticles obtained in step 4 of an embodiment of the present invention.

[0036] Figure 5 This is the serum stability result of the DNA origami structure of the functionalized magnetic nanoparticles obtained in step 4 of the embodiment of the present invention within two hours as determined by gel electrophoresis. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0038] A method for preparing a DNA origami structure based on functionalized magnetic nanoparticles comprises the following steps:

[0039] Step 1: Construct a DNA origami structure with a cavity through self-assembly. The DNA origami structure is composed of a scaffold chain and short chains that are complementary to its base pairs. The short chains include a first short chain extending into the cavity and a second short chain extending out of the cavity. The DNA origami structure has a three-dimensional tubular structure. The self-assembly conditions are as follows:

[0040] Keep warm at 65°C for 15 min;

[0041] The temperature was lowered to 50 °C and then to 40 °C at a rate of 0.1 °C per 39.6 min;

[0042] The temperature was lowered to 15°C at a rate of 1°C per 2.4 minutes.

[0043] Step 2: Connecting the thiol-functionalized first sequence to the first short chain through complementary base pairing, and chelating ferrous ions on the first sequence to obtain a thiol-modified DNA origami structure; the specific process is as follows:

[0044] Restore the first sequence;

[0045] Mixing the DNA origami structure obtained in step 1 with the reduced first sequence, and allowing the reaction to fully proceed to obtain a thiol-modified DNA origami structure;

[0046] The thiol-modified DNA origami structure is fully mixed with a ferrous ion solution to react, thereby obtaining a DNA origami structure chelating ferrous ions.

[0047] Step 3: reducing the ferrous ions chelated on the thiol-modified DNA origami structure obtained in step 2 to obtain a magnetic functionalized DNA origami structure loaded with Fe2O3 particles;

[0048] The restoration process is as follows:

[0049] The thiol-modified DNA origami structure obtained in step 2 is placed in a NaBH4 solution, and after sufficient reaction, a magnetically functionalized DNA origami structure can be obtained.

[0050] Step 4: Attach an antibody to the second short strand of the DNA origami structure obtained in Step 3 through base pairing to obtain the desired DNA origami structure. Attach the antibody using a biotin-streptavidin conjugation method.

[0051] The DNA origami structure has a three-dimensional tubular structure, with magnetic nanoparticles coupled inside and antibodies loaded on the outside.

[0052] Example

[0053] A method for preparing a DNA origami structure based on functionalized magnetic nanoparticles comprises the following steps:

[0054] Step 1: Construct a three-dimensional tubular DNA origami structure through a self-assembly method; the DNA origami structure is formed by a scaffold chain and short chains that are complementary to its bases; the short chains include a first short chain extending into the cavity, a second short chain extending out of the cavity, and a third short chain; the sequence of the first short chain is as shown in SEQ ID NO.1 to SEQ ID NO.46; the sequence of the second short chain is as shown in SEQ ID NO.47 to SEQ ID NO.102. The scaffold chain used is p7249, and the third short chain is the remaining complementary sequence of p7249. In this example, a DNA origami structure that meets the requirements is obtained through DNA design. The third short chain is set as needed, as long as it can complement the scaffold chain in addition to the first and second short chains, and the remaining sites are complementary to each other.

[0055] The distribution ratios of each group are as follows:

[0056] Short chain (including the first short chain, the second short chain, and the third short chain) 292 nM, volume 48.86 μl, scaffold chain 100 nM, volume 20.38 μl; 10X TAE buffer, volume 10 μl; 100 mM magnesium chloride, volume 10 μl; ultrapure water, volume 10.76 μl; the mixture, the final folded structure concentration is 21 nM, the total volume is 100 μl.

[0057] After the above components are mixed, self-assembly is carried out under the following conditions:

[0058] Heating stage: heating to 65°C and keeping warm for 15 min;

[0059] Slow annealing stage: the temperature was lowered to 50 °C, and then to 40 °C at a rate of 0.1 °C per 39.6 min;

[0060] Rapid annealing stage: After the temperature dropped to 40 °C, it was cooled to 15 °C at a rate of 1 °C per 2.4 min.

[0061] After annealing, the DNA was passed through a 100K ultrafiltration tube and centrifuged at 3000 rcf for 3 minutes, repeated five times to remove free sequences, thereby obtaining a purified and enriched DNA origami structure (nanobarrel).

[0062] The gel electrophoresis characterization results of the DNA origami structure obtained in this step are as follows Figure 1 As shown in the figure, it can be seen that the migration distance of the DNA origami structure is slightly lower than that of the corresponding photo (scaffold chain p7249), and there is no obvious polymer band, indicating that the DNA origami structure is successfully folded.

[0063] Step 2: First, the thiol-modified first sequence (thymine repeat sequence with thiol modification at the 3' end) is reduced with 200 mM TCEP to reduce the disulfide bonds therein to thiol groups. The first sequence and the first short chain sequence are complementary to each other.

[0064] The DNA origami structure purified in step 1 was mixed with the reduced 8X first sequence. The mixture was heated to 37°C for 15 minutes, then slowly cooled to room temperature and shaken at 200 rpm overnight to obtain the thiol-modified DNA origami structure nanobarrel-SH. The mixture was centrifuged through a 100K ultrafiltration tube at 3000 rcf for 3 minutes and washed five times with 1× TA / Mg²⁺ buffer (40 mM Tris, 20 mM glacial acetic acid, 12.5 mM magnesium chloride, pH 8.0) to remove most of the excess first sequence.

[0065] Mix 6 μl of 20 mM FeCl2·4H2O with 100 μl of 5 nM nanobarrel-SH and incubate at room temperature for 3 hours. The solution changes from clear to yellow, indicating that Fe²⁺ has been successfully chelated. Agarose gel electrophoresis is used for characterization and observation under UV light. The results are as follows: Figure 2 As shown in the figure, there are DNA bands in the solution after the reaction, but no bands in the precipitate, indicating that Fe²⁺ has been successfully chelated.

[0066] Step 3: Centrifuge using a 100K ultrafiltration tube at 3000 rcf for 3 minutes, repeating the centrifugation three times to remove the remaining FeCl2. Then, add 5 μl of 200 mM NaBH4 and react for 3 hours, producing a yellow precipitate, indicating that the Fe²⁺ is reduced to Fe2O3 clusters. After centrifugation, shake and disperse the mixture. Place the reaction mixture on a magnetic stand and magnetically absorb for 2 hours. Remove the supernatant to obtain a magnetically functionalized DNA origami structure. Characterization by agarose gel electrophoresis is shown in the following figure. Figure 3 As shown in the figure, the DNA structure is absent from the supernatant after magnetic adsorption, but DNA bands are present in the adsorption product, demonstrating that the structure has been successfully functionalized with the magnetic nanoparticles. However, the sample, likely due to its increased molecular weight after magnetic adsorption, remains largely within the gel pores.

[0067] Step 4: Attach the antibody to the second short strand of the DNA origami structure obtained in step 3 via biotin-streptavidin coupling.

[0068] Mix the biotin-labeled sequence 5'-AAT AAT AAT AAT AAT (which is complementary to the base pairing of the second short chain sequence) and streptavidin-labeled S100-β antibody at a ratio of 1.2:1 and incubate at room temperature for 4-6 hours to obtain the DNA sequence-labeled antibody.

[0069] Mix the labeled antibody with the magnetically functionalized DNA origami structure obtained in step 3 and shake at 200 rpm at 4°C overnight. Subsequently, ultrafiltration is used to remove unbound antibody, yielding the antibody-labeled nanobarrel, the desired DNA origami structure.

[0070] The DNA origami structure obtained in step 4 was characterized by transmission electron microscopy. Figure 4 As shown in the figure, it can be seen that dispersed, magnetic nanoparticles and antibody-labeled DNA origami structures were obtained.

[0071] Not only can antibodies be efficiently coupled to the nanobarrel surface, but when the antibody type needs to be changed, rapid switching can be achieved by simply replacing the streptavidin-labeled antibody, thus providing flexible antibody loading capacity.

[0072] The DNA origami structure obtained in step 4 was tested for serum stability as follows:

[0073] The structure was incubated with 20% serum at 37°C, and its serum stability was tested by agarose gel electrophoresis within 2 hours. Figure 5 As shown in the figure, it can be seen that the structure obtained by the present invention can maintain good structural integrity within 2 hours.

[0074] This invention leverages the adaptability of DNA origami structures to precisely load a fixed number of antibodies onto the same structure. The resulting DNA origami structure concentration can be accurately measured using a micro-UV spectrophotometer and relative molecular mass. This achieves a high degree of uniformity and stability, while also offering enhanced precision control capabilities, providing a more controllable carrier platform for related applications.

[0075] The DNA origami structures obtained in the present invention were used as magnetic beads to prepare a kit. The results of the comparison with existing kits are shown in Table 1.

[0076] Table 1. Comparison results of the kit to be evaluated (this example) and the comparative kit (existing)

[0077]

[0078] The present invention breaks the limitations of traditional applications. By introducing magnetic nanoparticles, it gives them controllable magnetic response characteristics, enabling them to have precise control and separation capabilities, and realize application scenarios beyond traditional DNA origami. Combining chain complementarity and metal chelation not only enhances the stability of magnetic nanoparticles on DNA origami, but also provides a more flexible functionalization method, realizing dual fixation measurement. The introduction of antibodies through the biotin-streptavidin interaction makes the entire structure more accurate and efficient in functional realization, thereby optimizing the immunoassay system and improving detection sensitivity and repeatability. Compared with traditional magnetic bead preparation methods, this strategy reduces the complex coupling and purification steps, while enhancing the stability of magnetic nanoparticles in biological detection environments, providing a new idea for the application of DNA origami technology in biological detection.

[0079] The present invention innovatively uses DNA molecules for the functionalization of magnetic nanoparticles, breaking through traditional applications, and using DNA origami to create precisely controllable biomagnetic beads. Acute cerebral stroke refers to a neurological disease in which the blood supply to the brain is suddenly interrupted, leading to softening and necrosis of brain tissue. It can lead to a variety of sequelae, and early diagnosis and symptomatic treatment are the key to improving prognosis. The ischemic and hypoxic damage to brain tissue worsens, accompanied by an increase in the necrotic part of the central nervous system. The secondary cerebral edema destroys the blood-brain barrier, and the cerebrospinal fluid S100-β protein can pass through the blood-brain barrier into the blood, or a large amount of S100-β is synthesized and released by glial cells and then passes through the damaged blood-brain barrier, causing a significant increase in the level of S100-β in the blood. Such results suggest that S100-β is involved in the pathogenesis of the brain and can also be used as an assessment indicator of the severity of the disease. In the embodiment, S100-β is loaded on a DNA origami structure and used as a nanomagnetic bead to prepare an S100-β protein detection kit. The resulting protein detection kit offers significant advantages. The programmability and precise spatial positioning capabilities of DNA origami enable highly quantitative and uniform antibody loading, thereby improving detection sensitivity and specificity. Incorporation of magnetic nanoparticles enables rapid separation and purification, simplifying the experimental process and improving detection efficiency. Furthermore, the nanoscale uniformity and quantitative control of the DNA origami structure ensure the stability and reproducibility of the kit. This technology is suitable for high-throughput screening and clinical applications, while also possessing versatility and scalability to meet diverse clinical needs.

Claims

1. A method for preparing a DNA origami structure based on functionalized magnetic nanoparticles, characterized in that: The following steps are involved: Step 1: Construct a DNA origami structure with a cavity by a self-assembly method; the DNA origami structure is formed by a scaffold chain and short chains that are complementary to the scaffold chain, the short chains including a first short chain extending into the cavity, a second short chain extending out of the cavity, and a third short chain; the sequence of the first short chain is such as SEQ ID NO.1 to SEQ ID NO.46; the sequence of the second short chain is such as SEQ ID NO.47 to SEQ ID NO.102; the scaffold chain is p7249, and the third short chain is the remaining complementary sequence of p7249; the self-assembly conditions are as follows: Keep warm at 65℃ for 15min; The temperature was lowered to 50°C, and then to 40°C at a rate of 0.1°C per 39.6 min; Cool down to 15°C at a rate of 1°C per 2.4 minutes; Step 2: Connecting the thiol-functionalized first sequence to the first short chain through base complementary pairing, and chelating ferrous ions on the first sequence to obtain a thiol-modified DNA origami structure; Step 3: oxidizing the ferrous ions chelated on the thiol-modified DNA origami structure obtained in step 2 to obtain a magnetic functionalized DNA origami structure loaded with Fe2O3 particles; Step 4: Connect the antibody to the second short chain of the DNA origami structure obtained in step 3 through base complementary pairing to obtain the desired DNA origami structure.

2. The method for preparing a DNA origami structure based on functionalized magnetic nanoparticles according to claim 1, characterized in that: The DNA origami structure in step 1 has a three-dimensional tubular structure.

3. The method for preparing a DNA origami structure based on functionalized magnetic nanoparticles according to claim 1, characterized in that: The specific process in step 2 is as follows: Restore the first sequence; Mixing the DNA origami structure obtained in step 1 with the reduced first sequence, and allowing the reaction to fully proceed to obtain a thiol-modified DNA origami structure; The thiol-modified DNA origami structure is fully mixed with a ferrous ion solution to react, thereby obtaining a DNA origami structure chelating ferrous ions.

4. The method for preparing a DNA origami structure based on functionalized magnetic nanoparticles according to claim 1, characterized in that: The restoration process in step 3 is as follows: The thiol-modified DNA origami structure obtained in step 2 is placed in a NaBH4 solution, and after sufficient reaction, a magnetically functionalized DNA origami structure can be obtained.

5. The method for preparing a DNA origami structure based on functionalized magnetic nanoparticles according to claim 1, characterized in that: In step 4, the antibody is linked by a biotin-streptavidin coupling method.

6. The DNA origami structure based on functionalized magnetic nanoparticles obtained by the preparation method according to any one of claims 1 to 5, characterized in that: The DNA origami structure has a three-dimensional tubular structure, is coupled with magnetic nanoparticles inside, and is loaded with antibodies on the outside.

7. The use of a DNA origami structure based on functionalized magnetic nanoparticles according to claim 6, characterized in that: The DNA origami structure is used to prepare biological magnetic beads.

8. The use of a DNA origami structure based on functionalized magnetic nanoparticles according to claim 7, characterized in that: The biological magnetic beads are used in a protein detection kit.

9. The use of a DNA origami structure based on functionalized magnetic nanoparticles according to claim 7, characterized in that: The antibody in the DNA origami structure is a specific antibody for the antigen to be detected.

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