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

Abstract

The invention relates to the field of DNA nanotechnology, and discloses a DNA origami structure based on functionalized magnetic nanoparticles, a preparation method and application, and the preparation method comprises the following steps: step 1, constructing a DNA origami structure with a cavity through a self-assembly method; the short chains in the DNA origami structure comprise a first short chain, a second short chain and a third short chain; 2, connecting a sulfydryl-functionalized first sequence to the first short chain, and chelating ferrous ions on the first sequence to obtain a sulfydryl-modified DNA origami structure; 3, ferrous ions chelated on the sulfydryl modified DNA origami structure are reduced, and a magnetic functionalized DNA origami structure internally loaded with Fe2O3 particles is obtained; and 4, connecting an antibody to a second short chain in the DNA origami structure through base complementary pairing to obtain the required DNA origami structure. According to the invention, DNA origami is combined with magnetic nanoparticles, efficient and controllable biological magnetic beads are obtained through dual immobilization and antibody modification, and a traditional application scene of DNA origami is broken through.

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] At present, early detection of diseases mainly relies on the detection of specific biomarkers, such as S100-β protein associated with brain damage, thrombomodulin and D-dimer associated with thrombosis. The detection of these biomarkers often uses magnetic nanoparticle adsorption based on magnetic beads. Magnetic beads are a special type of nano-micron material with a diameter usually ranging from several hundred nanometers to several microns, and have superparamagnetic properties. Under the action of an external magnetic field, the magnetic beads can quickly gather in the direction of the magnetic field, and quickly return to a dispersed state after the magnetic field is removed. When the surface of the magnetic beads is loaded with relevant antibodies, they can quickly bind to specific antigens, and then separate the magnetic beads from the substances to be detected by magnetic separation technology. By marking easily visible substances on antibodies or antigens, and then detecting changes in these markers, the binding reaction between antigens and antibodies can be indirectly reflected, thereby achieving accurate detection of trace antigens or antibodies. This method has significant advantages in improving detection sensitivity, specificity and ease of operation, and is widely used in early diagnosis of diseases. However, domestic magnetic bead carriers of uniform size still rely on imports, and the antigens loaded on the surface of the magnetic beads are usually not quantifiable. During the preparation of magnetic beads, repeated attempts and excessive incubation are often required to obtain magnetic beads with appropriate antigen loading. In this process, differences between carrier batches may significantly affect the accuracy of the test results.

[0003] DNA origami technology is a bio-nanotechnology based on DNA, which uses a circular single-stranded DNA and hundreds of precisely designed short-chain DNA to self-assemble into a predetermined nanostructure through program annealing under specific conditions. The programmability of this technology enables the DNA origami structure to be folded into a specific shape strictly according to 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 the 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, the nonspecific binding phenomenon may lead to an increase in background signals, reducing the detection sensitivity and accuracy; 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 surface of the magnetic beads 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 and affect 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, thus overcoming the limitations of traditional magnetic beads. Summary of the invention

[0006] In view of 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: Step 1: constructing a DNA origami structure with a cavity by a self-assembly method; the DNA origami structure is formed by a scaffold chain and a short chain complementary to its base pairing; the short chain includes a first short chain extending into the cavity, a second short chain extending out of the cavity, and a third short chain; Step 2: Connecting the first sequence functionalized with thiol groups to the first short chain through complementary base pairing, chelating ferrous ions on the first sequence, and obtaining a thiol-modified DNA origami structure; Step 3: Reduce the ferrous ions chelated on the thiol-modified DNA origami structure obtained in step 2 to obtain an internally loaded Fe 2 O 3 Magnetic functionalization of particles into DNA origami structures; Step 4: The desired DNA origami structure can be obtained by connecting the antibody to the second short chain in the DNA origami structure obtained in step 3 through base complementary pairing.

[0008] Furthermore, the DNA origami structure in step 1 has a three-dimensional tubular structure.

[0009] Furthermore, the self-assembly conditions in step 1 are as follows: Keep warm at 65°C for 15 min; The temperature dropped to 50 °C, and then dropped to 40 °C at a rate of 0.1 °C per 39.6 min; The temperature was lowered to 15 °C at a rate of 1 °C in 2.4 min.

[0010] Furthermore, the specific process in step 2 is as follows: Restore the first sequence; The DNA origami structure obtained in step 1 is mixed with the reduced first sequence, and after sufficient reaction, a thiol-modified DNA origami structure is obtained; The DNA origami structure modified with thiol is fully mixed with ferrous ion solution to react, so as to obtain the DNA origami structure chelating ferrous ions.

[0011] Furthermore, the restoration process in step 3 is as follows: The thiol-modified DNA origami structure obtained in step 2 was placed in NaBH 4 After sufficient reaction in the solution, a magnetic functionalized DNA origami structure can be obtained.

[0012] Furthermore, in step 4, the antibody is connected by a biotin-streptavidin coupling method.

[0013] A DNA origami structure based on functionalized magnetic nanoparticles has a three-dimensional tubular structure, with magnetic nanoparticles coupled inside and antibodies loaded outside.

[0014] An application of a DNA origami structure based on functionalized magnetic nanoparticles, wherein the DNA origami structure is used to prepare biological magnetic beads.

[0015] Furthermore, the biomagnetic beads are used in a protein detection kit.

[0016] Furthermore, the antibody in the DNA origami structure is a specific antibody for the antigen to be detected.

[0017] The beneficial effects of the present invention are: (1) 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; (2) The present invention precisely fixes magnetic nanoparticles on the DNA origami structure through chain complementation and metal chelation; (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

[0018] Figure 1 Schematic diagram of gel electrophoresis characterization of the DNA origami structure obtained in step 1 of the embodiment of the present invention.

[0019] 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.

[0020] 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.

[0021] Figure 4 This is a schematic diagram of the transmission electron microscopy characterization of the DNA origami structure of the functionalized magnetic nanoparticles obtained in step 4 of the embodiment of the present invention.

[0022] Figure 5 This is the serum stability result of the DNA origami structure gel electrophoresis detection of the functionalized magnetic nanoparticles obtained in step 4 of the embodiment of the present invention within two hours. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0024] A method for preparing a DNA origami structure based on functionalized magnetic nanoparticles comprises the following steps: Step 1: Construct a DNA origami structure with a cavity by self-assembly; the DNA origami structure is formed by a scaffold chain and a short chain complementary to its base pairing, the short chain includes 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: Keep warm at 65°C for 15 min; The temperature dropped to 50 °C, and then dropped to 40 °C at a rate of 0.1 °C per 39.6 min; The temperature was lowered to 15 °C at a rate of 1 °C in 2.4 min.

[0025] Step 2: Connect the first sequence functionalized with thiol groups to the first short chain through complementary base pairing, chelate ferrous ions on the first sequence, and obtain a thiol-modified DNA origami structure; the specific process is as follows: Restore the first sequence; The DNA origami structure obtained in step 1 is mixed with the reduced first sequence, and after sufficient reaction, a thiol-modified DNA origami structure is obtained; The DNA origami structure modified with thiol is fully mixed with ferrous ion solution to react, so as to obtain the DNA origami structure chelating ferrous ions.

[0026] Step 3: Reduce the ferrous ions chelated on the thiol-modified DNA origami structure obtained in step 2 to obtain an internally loaded Fe 2 O 3Magnetic functionalized DNA origami structures of particles; The restore process is as follows: The thiol-modified DNA origami structure obtained in step 2 was placed in NaBH 4 After sufficient reaction in the solution, a magnetically functionalized DNA origami structure can be obtained.

[0027] Step 4: The desired DNA origami structure is obtained by connecting the antibody to the second short chain of the DNA origami structure obtained in step 3 through base complementary pairing. The antibody is connected by biotin-streptavidin coupling method.

[0028] The DNA origami structure has a three-dimensional tubular structure, with magnetic nanoparticles coupled inside and antibodies loaded on the outside.

[0029] Example A method for preparing a DNA origami structure based on functionalized magnetic nanoparticles comprises the following steps: Step 1: construct a three-dimensional tubular DNA origami structure by a self-assembly method; the DNA origami structure is formed by a scaffold chain and a short chain complementary to its base pairing; the short chain includes a first short chain extending into the cavity, a second short chain extending out of the cavity, and a third short chain; wherein 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 used is p7249, and the third short chain is the remaining p7249 complementary sequence. This embodiment obtains a DNA origami structure that meets the requirements through DNA design. The third short chain is set as needed, as long as it can be complementary to the scaffold chain in addition to the first short chain and the second short chain, and the remaining sites are complementary to each other.

[0030] The distribution ratios of each group are as follows: 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; 10 X TAE buffer, volume 10 μl; 100 mM magnesium chloride, volume 10 μl; ultrapure water, volume 10.76 μl; the mixture obtained by mixing, the final folded structure concentration is 21 nM, the total volume is 100 μl.

[0031] After the above components are mixed, self-assembly is carried out under the following conditions: Heating stage: heating to 65 ℃ and keeping warm for 15 min; Slow annealing stage: the temperature dropped to 50 °C, and then dropped to 40 °C at a rate of 0.1 °C per 39.6 min; Rapid annealing stage: After the temperature dropped to 40 °C, it was cooled to 15 °C at a rate of 1 °C in 2.4 min.

[0032] After annealing, the DNA was filtered through a 100K ultrafiltration tube and centrifuged at 3000 rcf for 3 minutes, which was repeated five times to remove free sequences, thereby obtaining a purified and enriched DNA origami structure (nanobarrel).

[0033] 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.

[0034] Step 2: First, 200 mM TCEP is used to reduce the first sequence modified with thiol (thymine repeat sequence modified with thiol at the 3' end) to reduce the disulfide bonds therein to thiol. The first sequence and the first short chain sequence are complementary to each other in base pairing.

[0035] The DNA origami structure purified in step 1 was mixed with the reduced 8X first sequence, heated to 37°C and maintained 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. Centrifuge using a 100K ultrafiltration tube, 3000 rcf, centrifugation time 3 minutes, and rinsed with 1×TA / Mg²⁺ buffer (40 mM Tris, 20 mM glacial acetic acid, 12.5 mM magnesium chloride, pH 8.0), and repeated five times to remove most of the excess first sequence.

[0036] Add 6 μl of 20 mM FeCl 2 ·4H 2 O was mixed with 100 μl of 5 nM nanobarrel-SH and incubated at room temperature for 3 hours. The solution changed from clear to yellow, indicating that Fe²⁺ was successfully chelated. Agarose gel electrophoresis was 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.

[0037] Step 3: Centrifuge using a 100 K ultrafiltration tube at 3000 rcf for 3 minutes. Repeat the centrifugation three times to remove the remaining FeCl. 2 Then, 5 μl of 200 mM NaBH 4 The reaction was carried out for 3 hours and a yellow precipitate was produced, indicating that Fe²⁺ was reduced to Fe2 O 3 After centrifugation and oscillation, the reactants were placed in a magnetic rack and subjected to magnetic absorption for 2 hours. The supernatant was removed to obtain a magnetically functionalized DNA origami structure. Agarose gel electrophoresis was used for characterization. The results are shown in Figure 3 As shown in the figure, there is no DNA structure in the supernatant solution after magnetic adsorption, but there are DNA bands in the magnetic adsorption product, which proves that the structure has been successfully functionalized by magnetic nanoparticles. However, most of the sample exists in the gel pores because the molecular weight of the sample increases after magnetic adsorption.

[0038] Step 4: Connect the antibody to the second short strand in the DNA origami structure obtained in step 3 through biotin-streptavidin coupling.

[0039] The biotin-labeled sequence 5'-AAT AAT AAT AAT AAT (which is complementary to the base pairing of the second short chain sequence) and the streptavidin-labeled s100-β antibody were mixed at a ratio of 1.2:1 and incubated at room temperature for 4-6 hours to obtain a DNA sequence-labeled antibody.

[0040] The labeled antibody was mixed with the magnetically functionalized DNA origami structure obtained in step 3, and the mixture was shaken at 200 rpm at 4°C overnight. Subsequently, the unbound antibody was removed by ultrafiltration purification to obtain the antibody-labeled nanobarrel, i.e., the desired DNA origami structure.

[0041] 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 are obtained.

[0042] 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 having flexible antibody loading capacity.

[0043] The DNA origami structure obtained in step 4 was tested for its serum stability as follows: 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.

[0044] The present invention benefits from the variability of the DNA origami structure, and can accurately load a fixed number of antibodies on the same structure. The concentration of the obtained DNA origami structure can be accurately measured by a micro-ultraviolet spectrophotometer and relative molecular mass. It achieves a high degree of uniformity and stability, and also has stronger precision control capabilities, providing a more controllable carrier platform for related applications.

[0045] The DNA origami structure obtained in the embodiment of the present invention was used as magnetic beads to prepare a kit, and the results of comparison with the existing kit are shown in Table 1.

[0046] Table 1. Comparison results of the kit to be evaluated (this example) and the comparative kit (existing)

[0047] 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 realizes 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 to achieve dual fixed measurement. The introduction of antibodies through 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.

[0048] The present invention innovatively uses DNA molecules for the functionalization of magnetic nanoparticles, breaking through traditional applications, and DNA origami creates precise and controllable biomagnetic beads. Acute cerebral stroke refers to a neurological disease in which the blood supply to the brain is suddenly interrupted, resulting in softening and necrosis of brain tissue, which can lead to a variety of sequelae. Early diagnosis and symptomatic treatment are the key to improving prognosis. Brain tissue ischemic hypoxic damage is aggravated, accompanied by an increase in the necrotic part of the central nervous system, and secondary cerebral edema destroys the blood-brain barrier. 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 evaluation 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 a S100-β protein detection kit. The resulting protein detection kit has significant advantages. The programmability and precise spatial positioning capabilities of DNA origami make the antibody loading highly quantitative and uniform, thereby improving the sensitivity and specificity of the detection. The combination of magnetic nanoparticles can achieve rapid separation and purification, simplify the experimental process and improve the detection efficiency. In addition, 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, and is also versatile and scalable to meet different 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: constructing 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 complementary to the scaffold chain, wherein 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; Step 2: Connecting the first sequence functionalized with thiol groups to the first short chain through complementary base pairing, chelating ferrous ions on the first sequence, and obtaining a thiol-modified DNA origami structure; Step 3: reducing the chelated ferrous ions 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: The desired DNA origami structure can be obtained by connecting the antibody to the second short chain in the DNA origami structure obtained in step 3 through base complementary pairing.

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 self-assembly conditions in step 1 are as follows: Keep warm at 65°C for 15 min; The temperature dropped to 50 °C, and then dropped to 40 °C at a rate of 0.1 °C per 39.6 min; The temperature was lowered to 15 °C at a rate of 1 °C in 2.4 min.

4. 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; The DNA origami structure obtained in step 1 is mixed with the reduced first sequence, and after sufficient reaction, a thiol-modified DNA origami structure is obtained; The DNA origami structure modified with thiol is fully mixed with ferrous ion solution to react, so as to obtain the DNA origami structure chelating ferrous ions.

5. 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.

6. 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.

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

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

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

10. The use of a DNA origami structure based on functionalized magnetic nanoparticles according to claim 8, characterized in that: The antibody in the DNA origami structure is a specific antibody for the antigen to be detected.

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