Method for purifying product prepared from DNA origami nanostructure

Abstract

The invention belongs to the field of nanotechnology, and particularly relates to a method for purifying a DNA origami nanostructure preparation product, namely a method for purifying a plurality of DNA nanostructure monomers and high-order structures thereof by utilizing isodensity gradient ultra-high-speed centrifugation. DNA nanostructures of different sizes can be separated, and high-yield purification of DNA nanostructures across multiple orders of magnitude is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of nanotechnology, and specifically relates to a method for purifying a product prepared by a DNA origami nanostructure, namely, a method for purifying a variety of DNA nanostructure monomers and their high-order structures by utilizing isopycnic gradient ultrahigh-speed centrifugation. Background Art

[0002] Nanomaterials have a huge surface area and quantum size effects caused by their small size, which produce special properties different from ordinary materials, and thus have great application value in the fields of optics, electricity and magnetism. With the development of DNA self-assembly technology, DNA nanostructures have been expanded to a larger scale through hierarchical assembly technology. However, the traditional bottom-up upper assembly is often accompanied by a large number of substructures, resulting in a reduction in the yield of the target structure. The performance of nanomaterials is closely related to their size and morphology. For a long time, it has mainly relied on the improvement of synthesis methods to obtain relatively single-sized monodisperse nanoparticles as much as possible. However, due to the heterogeneity of synthesis conditions such as temperature field and concentration field during the synthesis process, it is difficult to obtain monodisperse nanoparticles. Therefore, how to obtain monodisperse nanostructures with single size and morphology is particularly important for the application of nanomaterials. Although the liquid phase synthesis method of inorganic nanomaterials has been developed more maturely, the controllable synthesis of monodisperse nanostructures is still limited to only a few systems. For most liquid-phase synthesized nanomaterials, subsequent separation and purification are essential. Existing separation methods mainly include chromatography, magnetic separation, ultrafiltration, centrifugal sedimentation, and electrophoresis, etc. However, these separation methods have great limitations for specific separation systems. For example, ultrafiltration is a common method for purifying DNA, but due to size limitations, it is not possible to purify the DNA superstructure. Column chromatography is also limited by size issues, and electrophoresis also has size limitations, resulting in a small amount of high-order structures that can be centrifuged and low recovery rates.

[0003] Therefore, it is imperative to establish an efficient and universal separation method. Density gradient centrifugation is an accurate experimental separation method established due to the continuous development of molecular biology. It was initially widely used in the separation and purification of biological macromolecules such as proteins, mitochondria, lysosomes, plant viruses, RNA, and DNA. Nanomaterials are similar to biological macromolecules in scale, so density gradient centrifugation is also applicable to the separation and purification of nanomaterials. Isopycnic gradient ultracentrifugation utilizes the difference in glycerol density and ultra-high centrifugal force to separate DNA nanostructures of different sizes, and has a high recovery rate and small loss while being not limited by size. It can also increase the centrifugal capacity by multiple orders of magnitude by replacing accessories, making the large-scale application of DNA nanostructures possible. Due to the lack of a complete system, it is used to integrate the separation of various DNA nanostructures. For example, various DNA origami nanomaterials targeted by this application, nanoparticles of different densities, due to different sedimentation coefficients, will produce different centrifugal results under different centrifugal conditions. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems of low recovery rate, size limitation, small scale and the like of the existing nanostructure preparation product purification method, and provide a purification method of DNA origami nanostructure preparation product using isopycnic gradient ultracentrifugation, and the DNA origami nanostructure preparation product to be purified contains DNA origami nanomonomer structure and its high-order structure, i.e., DNA origami nanostructures with different polymerization numbers, including DNA origami rectangular structure, the size of DNA origami rectangular structure is at least 1000×70nm, and the number of base pairs ranges at least 75000bp, and the high-order structure refers to the N-polymer structure formed by the polymerization of DNA origami nanomonomers, and N is not less than 2; the isopycnic gradient ultracentrifugation utilizes the difference in linear glycerol density and ultrahigh centrifugal force to separate DNA nanostructures of different sizes, and realizes high-yield purification of DNA nanostructures across multiple orders of magnitude. At the same time, the separation degree of ultracentrifugation is further improved, from 7249 base pairs to 75000 base pairs. The ultracentrifugation method disclosed in the present invention is not only efficient and multifunctional, but also scalable, and is an ideal solution for large-scale DNA nanostructure preparation.

[0005] To achieve this purpose, the present invention adopts the following technical solutions: Figure 5 As shown, the present invention provides a method for purifying a product prepared by a DNA origami nanostructure, the purification method comprising the steps of:

[0006] Step S1, preparing the DNA origami nanostructure product to be purified into an aqueous solution with a concentration of 2 nM;

[0007] Step S2, such as Figure 5As shown in Figure a, glycerol is prepared into glycerol buffer solutions with volume percentages of 50%, 55%, 60%, 65% and 70%, respectively. The glycerol buffer solution is composed of three components: glycerol, buffer solution and water. After the glycerol buffer solution is shaken and evenly mixed, it needs to be left standing overnight at room temperature of 25°C to 35°C because it contains a large number of bubbles. Then, equal volumes of solutions are taken in order from high concentration to low concentration to prepare a gradient solution, which is left standing overnight at 4°C to make the glycerol stratification more obvious.

[0008] Step S3, such as Figure 5 As shown in Figure b, the dispersion obtained in step S1 is slowly added to the top layer of the gradient solution obtained in step S2, and ultra-high-speed centrifugation is performed. Then, according to the number of portions of the gradient solution, the mixed solution after centrifugation is slowly aspirated in equal parts, and finally DNA nanoorigami structures with different polymerization numbers are obtained.

[0009] As a specific embodiment of the present application, the DNA origami nanostructure is a DNA origami rectangular structure, the size of the DNA origami rectangular structure is 1000×70nm, and the number of base pairs is 75000bp, the DNA origami nanostructure to be purified is a mixed DNA origami rectangular structure with a polymerization number of ten or less, the ultra-high speed centrifugal force in step S3 is set to 320,000rcf, the centrifugation time is 1.5h, and the rotor model is SW 55TI.

[0010] Preferably, the buffer is 10×TAE-Mg 2+ Buffer.

[0011] Compared with the prior art, the present invention has the following beneficial effects: the purification method of the DNA origami nanostructure preparation product provided by the present invention broadens the size of the separable DNA nanostructure, from the DNA rectangular origami decamer structure to the DNA rectangular origami monomer, and realizes the high-yield purification of DNA nanostructures spanning multiple orders of magnitude; when purifying DNA nanostructures, the present invention, especially when purifying high-order structures of DNA nanostructures, exhibits excellent recovery effect and low loss rate; the present invention can also increase the amount of purifiable samples to hundreds of milliliters at one time by replacing the centrifuge rotor assembly accessories. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is an atomic force microscope morphology characterization image of the DNA rectangular origami decamer before purification in Example 1, and the scale length in the image is 500 nm;

[0013] Figure 2 This is the atomic force microscope morphology characterization diagram of the DNA rectangular origami decamer purified by agarose gel electrophoresis in Comparative Example 1, and the scale length in the figure is 500 nm;

[0014] Figure 3This is an atomic force microscope morphology characterization image of the purified DNA rectangular origami decamer in Example 2, and the scale length in the image is 500 nm;

[0015] Figure 4 This is a characterization diagram of agarose gel electrophoresis analysis of the purified DNA rectangular origami decamer in Example 2;

[0016] Figure 5 This is a schematic diagram of the operation flow of the purification method of the DNA origami nanostructure preparation product of the present invention;

[0017] Figure 6 Schematic diagram of the design of the connecting chain of the DNA rectangular origami decamer according to an embodiment of the present invention. DETAILED DESCRIPTION

[0018] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0019] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0020] Buffers were purchased from Bio-Technology Company;

[0021] Sequence design of ten kinds of DNA origami for making decamer samples: each DNA rectangular origami is composed of a template assembled from an M13mp18 template chain and a DNA short chain, and the DNA short chain used to make the DNA rectangular origami includes a DNA staple chain and a connecting chain. The sequences of the above-mentioned M13mp18 template chain and DNA short chain refer to the corresponding M13mp18 template chain and DNA staple chain disclosed in the "Supporting information" of the document "Tuning the structural asymmetries of three-dimensional gold nanorod assemblies" with DOI number 10.1039 / c5cc05295e. The only difference is that the DNA staple chain used to assemble each DNA rectangular origami of this embodiment does not include 24 chains numbered 100-111 and 205-216 in the document, and the remaining DNA staple chains are the same as those disclosed in the document; and accordingly, a total of 18 groups of connecting chains are designed for the ten rectangular DNA origami R1-R10, with 6 connecting chains in each group, such as Figure 6As shown, the connecting chains are assembled on the rectangular DNA origami structure to form sticky ends, wherein the sticky ends of the RA group and the Ra group are complementary paired, the sticky ends of the RB group and the Rb group are complementary paired, the sticky ends of the RC group and the Rc group are complementary paired, the sticky ends of the RD group and the Rd group are complementary paired, the sticky ends of the RE group and the Re group are complementary paired, the sticky ends of the RF group and the Rf group are complementary paired, the sticky ends of the RG group and the Rg group are complementary paired, the sticky ends of the RH and Rh groups are complementary paired, and the sticky ends of the RI group and the Ri group are complementary paired; and the sticky ends of the RA group, the RB group, the RC group, and the RD group are complementary paired. The assembly sites of the six connecting chains of any group among the RE group, RF group, RG group, RH group, and RI group correspond to the sites numbered 100, 101, 104, 105, 108, and 109 in the literature, respectively, which is equivalent to replacing these six chains in the literature; similarly, the assembly sites of the six connecting chains of any group among the Ra group, Rb group, Rc group, Rd group, Re group, Rf group, Rg group, Rh group, and Ri group correspond to the sites numbered 216, 215, 212, 211, 208, and 207 in the literature, respectively, which is equivalent to replacing these six chains in the literature.

[0022] Among them, RA-Ra, RB-Rb, RC-Rc, RD-Rd, RE-Re, a total of 60 chains, refer to the sequences disclosed in the patent document of this group with publication number CN116227589A, entitled A DNA nanocomputing element, preparation method and application thereof, as shown in SEQ ID NO: 14-SEQ ID NO: 73, and the sequences of the remaining four groups of connecting chains RF-Rf, RG-Rg, RH-Rh, RI-Ri, a total of 48 connecting chains, are shown in SEQ ID NO: 1 to SEQ ID NO: 48 in the sequence list;

[0023] Example 1: Preparation of a DNA-containing rectangular origami decamer sample to be purified

[0024] Step S11, M13mp18 template strand and DNA short strand, DNA short strand includes: DNA staple strand, connecting strand, the M13mp18 template strand and DNA short strand are mixed in 1×TAE / Mg 2+ In the buffer, the total volume after mixing was 100 μL, in which the concentration of the template strand was 5 nM, the DNA short strands of each sequence were added in equal amounts and in excess, and the concentration of the DNA short strands was 50 nM;

[0025] Step S12, using a gradient PCR instrument to slowly anneal the mixture containing the template strand and the DNA short strand in step S11, the annealing conditions are: starting temperature 95°C, maintained for 3 minutes, end temperature 25°C, each 1°C is a gradient, each gradient stays for 100 seconds, and annealing from 95°C to 25°C within two hours;

[0026] Step S13, taking out the sample and centrifuging it in a 100 kDa centrifuge tube to remove excess DNA short chains; the centrifugation conditions are: adding 300 μL 1×TAE-Mg 2+ Buffer, centrifuged at 3000 rcf / min for 10 min, repeated centrifugation 3 times; ten purified DNA origami nanomonomer structures were obtained, and finally collected and measured by a micro-UV spectrophotometer to have a concentration of 2 nM.

[0027] The ten kinds of DNA origami nanomonomer structure samples purified in step S13 were mixed evenly in an equal molar ratio. The system was 250 μL, the concentration of the DNA rectangular origami monomer was 2 nM, and put into PCR. The starting temperature was 45°C, maintained for 5 min, then dropped to 37°C, with a gradient of 0.1°C for 99 cycles, then dropped to 25°C, with a gradient of 0.1°C for 99 cycles, and finally maintained at 25°C. The assembly was completed after 12 hours.

[0028] like Figure 1 Shown is the AFM characterization of the DNA-containing rectangular origami polymer sample to be purified after assembly. The AFM characterization results show that the DNA origami decamer is fully assembled and that polymer structures such as DNA origami monomers, dimers, and trimers exist in the sample at the same time.

[0029] Example 2 Ultracentrifugation purification

[0030] Step S21, the DNA-containing rectangular origami polymer sample to be purified prepared in Example 1 is dispersed by oscillation;

[0031] Step S22, according to the formula shown in Table 1, prepare linear glycerol into glycerol buffer solutions with volume percentages of 50%, 55%, 60%, 65% and 70%, respectively, shake well, and let stand at room temperature 25°C overnight, until the solution contains a large number of bubbles; then, take equal volumes of solution in order from high concentration to low concentration and add them to 5mL centrifuge tubes, with a volume of 667uL added to each layer, let stand at 4°C overnight, and obtain a glycerol gradient solution;

[0032] Step S23, add 200uL of the assembly product obtained in step S2 to the top layer of the centrifuge tube containing the glycerol gradient solution in step S22, place the centrifuge tube in an ultra-high speed centrifuge, and after height adjustment, select a centrifugal force of 320,000rcf and a centrifugal time of 1.5h (SW 55TI).

[0033] Table 1

[0034]

[0035] Comparative Example 1:

[0036] In order to test the feasibility of the method, the steps of Comparative Example 1 differ from those of Example 2 only in that, in step S23, a centrifugal force of 250,000 rcf and a centrifugation time of 1.5 h are selected for purification, and the remaining steps and raw materials are the same: 200 uL, 2 nM sample purification, and the DNA-containing rectangular origami polymer sample to be purified prepared in Example 1 is also used.

[0037] Test Example 1 Electrophoresis Characterization of Purified Samples

[0038] 100uL of the centrifuged product obtained in Example 2 was extracted from top to bottom, and a total of 34 layers were extracted. 20uL of each layer was extracted for 0.5% agarose gel electrophoresis characterization and compared with the product before purification. Figure 4 As shown, it can be seen that after purification, the target product and other by-products are separated in a step-like manner.

[0039] Test Example 2 AFM Characterization of Purified Samples

[0040] The centrifuged products obtained in Example 2 and Comparative Example 1 were combined by selecting the corresponding number of layers according to the bands shown in Test Example 1. Since the initial sample concentration was low, it was added to a 100 kDa centrifuge tube for concentration. The centrifugal force was less than 2000 rcf. At the same time, since the glycerol concentration used in the DNA origami polymer was high, 1×TAE / MgCl2 was added. 2+ The obtained centrifuged product was characterized by AFM gas phase. Figure 2 This is the AFM characterization image of Comparative Example 1, Figure 3 This is the AFM characterization diagram of Example 2, compared with Figure 2 and Figure 3 It can be seen from the results that the decamer purification effect obtained in Example 2 is significantly better than the purification effect obtained under the conditions of Comparative Example 1.

[0041] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for purifying a product prepared by a DNA origami nanostructure, the method comprising the steps of: Step S1, preparing the DNA origami nanostructure product to be purified into an aqueous solution with a concentration of 2 nM; Step S2, preparing glycerol into glycerol buffer solutions with volume percentages of 50%, 55%, 60%, 65% and 70% respectively, wherein the glycerol buffer solution is composed of three components: glycerol, buffer solution and water; after the glycerol buffer solution is shaken and evenly mixed, it is allowed to stand overnight at room temperature of 25° C. to 35° C., and then equal volumes of solutions are taken in order from high concentration to low concentration to prepare a gradient solution, and the solution is allowed to stand overnight at 4° C.; Step S3, slowly adding the dispersion obtained in step S1 to the top layer of the gradient solution obtained in step S2; after ultra-high speed centrifugation, the centrifuged mixed solution is slowly aspirated in equal parts according to the number of portions of the gradient solution, and finally DNA nano-origami structures with different polymerization numbers are obtained.

2. The method for purifying a DNA origami nanostructure preparation product according to claim 1, characterized in that: The DNA origami nanostructure is a DNA origami rectangular structure.

3. The method for purifying a DNA origami nanostructure preparation product according to claim 2, characterized in that: The size of the DNA origami rectangular structure is 1000×70 nm, and the number of base pairs is 75,000 bp. The DNA origami nanostructure to be purified is a mixed DNA origami rectangular structure with a polymerization degree of ten or less.

4. The method for purifying a DNA origami nanostructure preparation product according to claim 3, characterized in that: In step S3, the ultra-high-speed centrifugal force is set to 320,000 rcf, the centrifugation time is 1.5 h, and the rotor model is SW 55TI.

5. The method for purifying a DNA origami nanostructure preparation product according to claim 4, characterized in that: The buffer is 10×TAE-Mg 2+ Buffer.

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