Method for preparing structure-dynamically controllable nanoparticle superlattice based on DNA origami crystal template

Abstract

The present invention discloses a preparation method of a nanostructure-dynamically controllable nanoparticle superlattice based on a DNA origami crystal template, comprising the following steps: (1) synthesizing a variety of DNA origami framework structures with specific ligation ability and selective loading ability; (2) modifying gold nanoparticles with functionalized thiol DNA strands on the surface; (3) synthesizing a multi-component co-crystalline DNA origami crystal substrate loaded with gold nanoparticles at specific sites by a one-step method; (4) regulating the order and timing of adding different DNA segments in the washed substrate, supplemented by constant temperature heat treatment, to obtain a nanostructure-dynamically controllable nanoparticle superlattice based on the DNA origami crystal template. Based on the DNA origami co-crystallization technology, the present invention realizes the controllable and on-demand transformation between various nanoparticle superlattices without breaking the original template.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing a structurally dynamically controllable nanoparticle superlattice based on a DNA origami crystal template. Background Art

[0002] DNA origami is a nanotechnology that uses hundreds of artificially designed short chains to assist in folding a long DNA chain to form a specific structure. The structure prepared by it has excellent addressability and programmability. In recent years, using this structural advantage to self-assemble DNA origami units to form templates, thereby performing multi-dimensional orderly assembly of nano-scale guest substances, has become a research hotspot in the field of DNA nanotechnology. The polyhedral framework structure prepared by DNA origami has great potential in constructing DNA origami templates due to its good guest carrying capacity and specific assembly ability. At present, researchers have successfully used origami frameworks in the shape of octahedrons, cubes, tetrahedrons, etc. to form templates, guide nanoparticles to crystallize together, and construct a variety of nanoparticle superlattices such as simple cubic, face-centered cubic, and diamond structures (Nature Mater., 2020, 19, 789–796). Subsequently, Professor Tian Ye's research group made full use of the sequence recognition characteristics of DNA and developed a method to co-crystallize origami frameworks of different shapes, successfully realizing the multi-co-crystallization of origami monomers (J.Am.Chem.Soc., 2020, 142, 21336-21343), laying the foundation for constructing origami templates with more diverse structures. However, the specific structure of the DNA origami templates and nanoparticle superlattices obtained in existing studies has been determined at the monomer synthesis stage, and the structure cannot change after crystal synthesis. If you want to obtain a new type of nanoparticle superlattice, you need to start from scratch and modify the corresponding monomers.

[0003] This feature greatly reduces the utilization rate of origami templates. At present, researchers have tried to modify the lattice parameters of superlattices after crystal synthesis, such as replacing the monomer connection part with an i-motif or hairpin structure that can change configuration with corresponding stimuli (Angew. Chem. Int. Ed., 2022, 61, e202208290; J. Am. Chem. Soc., 2023, 145, 3978-3986). These results confirm the possibility of manipulating nanoparticle superlattices without destroying the origami template, but it is essentially manipulating the origami template and is limited to the change of lattice parameters, and cannot achieve dynamic changes in crystal types. Therefore, it is necessary to develop a method for dynamically controlling and on-demand transformation of superlattice structure types by manipulating nanoparticles under a fixed DNA origami template, thereby greatly improving the application potential of DNA origami templates in constructing multi-type nanoparticle superlattices. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing a nanostructured dynamic controllable nanoparticle superlattice based on a DNA origami crystal template without damaging the DNA origami template.

[0005] To achieve the above application object, the technical solution adopted in this application is as follows: A method for preparing a nanostructured dynamic controllable nanoparticle superlattice based on a DNA origami crystal template of the present invention includes the following steps:

[0006] (1) Synthesize a variety of DNA origami framework structures: DNA origami consists of M13mp18 long chains, staple chains, sticky end chains, inner chains and pre-sealed chains; Mix the M13mp18 long chains, hundreds of staple chains, sticky end chains, inner chains and pre-sealed chains in a molar ratio of 1:10:10:7.5:30 in 1XTAE, 12.5 mM Mg(CH3COO)2 solution, and the mixture undergoes a gradient cooling program of 90-20 °C in PCR for 22-24 h, and finally DNA origami frameworks with a concentration of 10 nM are obtained.

[0007] The nucleotide sequences of the inner chains are as shown in SEQ NO: 1, 2, 3, 4, 5…16.

[0008] The staple chains are divided into three sets, namely staple chain-1, staple chain-2 and staple chain-3;

[0009] Staple chain-1: The first set of staple chains, a total of 116 roots, removing the four numbered 100 / 101 / 111 / 117, and the remaining numbers are the same as those in part a of the DNA sequences in the article;

[0010] Staple chain-2: The second set of staple chains, a total of 140 roots, removing the four numbered 121 / 129 / 138 / 142, and the remaining numbers are the same as those in part b of the DNA sequences in the article;

[0011] Staple chain-3: The third set of staple chains, a total of 128 roots, removing the four numbered 111 / 114 / 122 / 125, and the remaining numbers are the same as those in part c of the DNA sequences in the article;

[0012] The sticky-end strands are divided into four sets. Sticky-end strand - 1: It is identical to R-oct-1 in DNA sequences g of the article; Sticky-end strand - 2: It is identical to E-oct-1 in DNA sequences g of the article; Sticky-end strand - 3: It is identical to P-oct-1 in DNA sequences g of the article; Sticky-end strand - 4: It is identical to P-oct-2 in DNA sequences g of the article;

[0013] The sequences of the staple strands and sticky-end strands can be found in DNA sequence parts a, b, c, g in the supplementary information of Sci.Adv., 2022, 8, eadc9755. The sequence information of the inner strands and pre-sealed strands is shown in the sequence listing;

[0014] (2) Modify the functionalized thiol DNA strand on the surface of 10 nm gold nanoparticles: First, use TCEP to reduce the DNA strand modified with a disulfide bond at one end to expose the thiol terminus. Subsequently, purify it with a G-25 size-exclusion centrifugal column. Then, mix the purified thiol DNA strand with 10 nm gold nanoparticles at a molar ratio of 300:1. The reaction time at room temperature is 1.5 - 2 h. Subsequently, add an appropriate amount of PB buffer to make its final concentration 10 mM. Then, slowly add 2 M NaCl solution successively to make the final concentration of NaCl 0.3 M. The mixed solution is placed at room temperature and rotated for reaction for 18 - 24 h. Finally, purify the reacted gold nanoparticles by high-speed centrifugation multiple times to remove the excess thiol DNA strand in the solution. The modified gold nanoparticles are dispersed in 0.1 M PBS buffer and stored statically at 4 °C;

[0015] (3) Prepare a DNA origami crystal substrate loaded with gold nanoparticles at specific sites in one step: Mix the origami framework prepared in step (1) with the modified gold nanoparticles in step (2) at a molar ratio of 1:0.9 - 1:1. Then, add an excessive poly-A sequence. After dispersing evenly, anneal it twice in a crystal incubator in the temperature range of 50 - 20 °C to obtain a DNA origami crystal substrate. The nucleotide sequence of poly-A is as shown in SEQ NO: 25.

[0016] (4) Wash the origami crystal substrate to remove the excess DNA segments and gold nanoparticles: Wash the substrate obtained in step (3) multiple times with 1XTAE and 12.5 mM Mg(CH3COO)2 solution;

[0017] (5)Regulate the order and timing of the addition of different displacement strands in the washed substrate, supplemented by constant temperature heat treatment, to achieve the conversion of the crystal structure of the nanoparticle superlattice between multiple states: According to the preset crystal structure change route, add the corresponding displacement strands to the washed substrate at an initial ratio of 1:5 for the molar ratio of each inner strand to the displacement strand. The displacement strands are divided into two types, R and P, where the R strand was called the pre-sealing strand when synthesizing various DNA origami framework structures in step (1); the nucleotide sequences of the displacement strands are as shown in SEQ NO: 17, 18, 19, 20, 21…24; then let it stand at a constant temperature.

[0018] Furthermore, in step (1), the staple strand, sticky end strand, inner strand, and pre-sealing strand should be appropriately selected according to the shape, connection ability, and loading ability of the origami framework, and a total of eight different origami monomers are involved.

[0019] Furthermore, in step (3), the origami crystal substrates are divided into two categories, the initial state and the intermediate state. Regardless of which category, four different DNA origami frameworks are required. All monomers of the initial state substrate can load nanoparticles, and the intermediate state substrate only loads nanoparticles at specific sites. The addition amount of gold nanoparticles is calculated according to the origami monomers with loading ability.

[0020] Even further, in step (3), the poly-A sequence needs to be added in excess at a ratio of 1:10 for the molar ratio of each sticky end strand: poly-A sequence.

[0021] Furthermore, in step (4), after washing the substrate, each tube of the substrate needs to be made up to a volume of 20 ul for subsequent use.

[0022] Furthermore, in step (5), after adding the displacement strands, the origami crystal needs to be allowed to stand at a constant temperature of 37 °C or 46 °C for 24 h.

[0023] Even further, in step (5), after the heat treatment is completed, the crystal needs to be washed with a 1XTAE, 12.5 mM Mg(CH3COO)2 solution. The washed crystal is stored at 4 °C.

[0024] Beneficial effects: The present invention realizes the manipulation of the nanoparticles therein without destroying the DNA origami template, enabling the nanoparticle superlattice to transform between multiple states in a dynamically controllable manner. This method is simple to operate and has strong scalability, and also has application potential on more complex DNA origami templates.

[0025] Compared with the prior art, the present invention has the following advantages: (1) The dynamic transformation of the nanoparticle superlattice structure achieved by the present invention is carried out after the crystal has been formed, and the DNA origami template is not damaged during the transformation process.

[0026] (2) The dynamic transformation of the nanoparticle superlattice structure realized by the present invention only requires the position regulation of the nanoparticles in the DNA origami template, and various types of nanoparticle superlattices can be obtained without the need to design and modify the monomers from scratch. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 It is the structural transformation roadmap of Example 1 of the present invention.

[0029] Figure 2 It is the small-angle X-ray scattering characterization result diagram of each node of Example 1 of the present invention.

[0030] Figure 3 It is the structural transformation roadmap of Example 2 of the present invention.

[0031] Figure 4 It is the scanning electron microscope characterization result diagram of the superlattice after the crystal structure transformation of Example 2 of the present invention.

[0032] Figure 5 It is the technical roadmap for manipulating the nanoparticle superlattice on the fixed DNA origami template of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] In order to make the technical problems to be solved, technical solutions and beneficial effects of the present application clearer, the following further describes the present application in detail with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0034] The following examples are helpful for further understanding the present invention, but the present invention is not limited thereto.

[0035] The first aspect of the present invention provides a preparation method of a structurally dynamically controllable nanoparticle superlattice based on a DNA origami crystal template, including the following steps:

[0036] In step (1), multiple DNA origami framework structures are synthesized: The M13mp18 long chain, hundreds of staple strands, sticky end strands, inner strands, and pre-sealing strands are mixed in a solution of 1XTAE and 12.5 mM Mg(CH3COO)2 at a molar ratio of 1:10:10:7.5:30. The mixture undergoes a gradient cooling program from 90 to 20 °C in a PCR for 22 h, and finally DNA origami frameworks with a concentration of 10 nM are obtained; The DNA origami is composed of the M13mp18 long chain, staple strands, sticky end strands, inner strands, and pre-sealing strands;

[0037] The nucleotide sequences of the inner strands are as shown in SEQ NO: 1, 2, 3, 4, 5…16;

[0038] The staple strands are divided into three sets, namely staple strand - 1, staple strand - 2, and staple strand - 3;

[0039] Staple strand - 1: The first set of staple strands is 116 in number, with four strands numbered 100 / 101 / 111 / 117 removed, and the remaining numbers are the same as those in part a of the DNA sequences in the article;

[0040] The sticky end strands are divided into four sets, sticky end strand - 1: It is the same as R - oct - 1 in part g of the DNA sequences in the article; The staple strands, sticky end strands, inner strands, and pre-sealing strands should be appropriately selected according to the shape, connection ability, and loading ability of the origami framework, and a total of eight different origami monomers are involved.

[0041] In some embodiments, the DNA origami is composed of the M13mp18 long chain, staple strands, sticky end strands, inner strands, and pre-sealing strands; The replacement strands are divided into two types, R and P, where the R strand is called the pre-sealing strand when synthesizing multiple DNA origami framework structures in step (1);

[0042] Staple strand - 2: The second set of staple strands is 140 in number, with four strands numbered 121 / 129 / 138 / 142 removed, and the remaining numbers are the same as those in part b of the DNA sequences in the article;

[0043] Sticky end strand - 2: It is the same as E - oct - 1 in part g of the DNA sequences in the article; Sticky end strand - 3: It is the same as P - oct - 1 in part g of the DNA sequences in the article; Sticky end strand - 4: It is the same as P - oct - 2 in part g of the DNA sequences in the article.

[0044] In some embodiments, the DNA origami is composed of M13mp18 long chains, staple chains, sticky-end chains, inner chains, and pre-sealing chains; the displacement chains are divided into two types, R and P, where the R chain is called the pre-sealing chain when synthesizing various DNA origami framework structures in step (1); the nucleotide sequences of the inner chains are as shown in SEQ NO: 1, 2, 3, 4, 5…16.

[0045] Staple chain - 3: The third set of staple chains consists of 128 strands. Four strands numbered 111 / 114 / 122 / 125 are removed, and the remaining numbers are consistent with part c of the DNA sequences in the article.

[0046] Sticky-end chain - 3: It is consistent with P-oct-1 in part g of the DNA sequences in the article; Sticky-end chain - 4: It is consistent with P-oct-2 in part g of the DNA sequences in the article.

[0047] In some embodiments, in step (2), a functionalized thiol DNA chain is modified on the surface of 10nm gold nanoparticles: First, TCEP is used to reduce the DNA chain modified with a disulfide bond at one end to expose the thiol terminus, and then it is purified using a G-25 size exclusion centrifugal column. Next, the purified thiol DNA chain is mixed with 10nm gold nanoparticles at a ratio of 300:1, and the reaction time at room temperature is 1.5h. Subsequently, an appropriate amount of PB buffer is added to make its final concentration 10mM. Then, 2M NaCl solution is slowly added successively to make the final concentration of NaCl 0.3M. The mixed solution is placed in a rotary reaction at room temperature for 20h. Finally, the reacted gold nanoparticles are purified by high-speed centrifugation multiple times to remove the excess thiol DNA chain in the solution. The modified gold nanoparticles are dispersed in 0.1M PBS buffer and stored statically at 4°C.

[0048] In some embodiments, in step (2), a functionalized thiol DNA chain is modified on the surface of 10nm gold nanoparticles: First, TCEP is used to reduce the DNA chain modified with a disulfide bond at one end to expose the thiol terminus, and then it is purified using a G-25 size exclusion centrifugal column. Next, the purified thiol DNA chain is mixed with 10nm gold nanoparticles at a ratio of 300:1, and the reaction time at room temperature is 1.8h. Subsequently, an appropriate amount of PB buffer is added to make its final concentration 10mM. Then, 2M NaCl solution is slowly added successively to make the final concentration of NaCl 0.3M. The mixed solution is placed in a rotary reaction at room temperature for 20h. Finally, the reacted gold nanoparticles are purified by high-speed centrifugation multiple times to remove the excess thiol DNA chain in the solution. The modified gold nanoparticles are dispersed in 0.1M PBS buffer and stored statically at 4°C.

[0049] In some embodiments, in step (2), a functionalized thiol DNA strand is modified on the surface of 10 nm gold nanoparticles: First, TCEP is used to reduce the DNA strand modified with a disulfide bond at one end to expose the thiol terminus. Subsequently, purification is performed using a G-25 size exclusion centrifugal column. Then, the purified thiol DNA strand and 10 nm gold nanoparticles are mixed at a ratio of 300:1, and the reaction time at room temperature is 2 h. Subsequently, an appropriate amount of PB buffer is added to make its final concentration 10 mM. Then, 2 M NaCl solution is slowly added successively to make the final concentration of NaCl 0.3 M. The mixed solution is placed at room temperature and rotated for reaction for 20 h. Finally, the gold nanoparticles after the reaction are purified by high-speed centrifugation multiple times to remove the excess thiol DNA strand in the solution. The modified gold nanoparticles are dispersed in 0.1 M PBS buffer and stored statically at 4°C;

[0050] In step (3), a DNA origami crystal substrate loaded with gold nanoparticles at specific sites is prepared by a one-step method: The origami framework prepared in step (1) and the gold nanoparticles modified in step (2) are mixed at a molar ratio of 1:0.9. Subsequently, an excessive amount of poly-A sequence is added, and the nucleotide sequence of poly-A is as shown in SEQ ID NO: 25. After being evenly dispersed, it is placed in a crystal incubator and annealed twice within the temperature range of 50 - 20°C to obtain a DNA origami crystal substrate;

[0051] The DNA origami crystal substrates are divided into two categories: the initial state and the intermediate state. Regardless of which category, four different DNA origami frameworks are required. All monomers of the substrate in the initial state can be loaded with nanoparticles, and the substrate in the intermediate state is only loaded with nanoparticles at specific sites. The addition amount of gold nanoparticles is calculated based on the origami monomers with loading capacity. An excessive amount of poly-A sequence needs to be added at a molar ratio of each sticky end strand: poly-A sequence of 1:10. After washing the substrate, each tube of the substrate needs to be made up to 20 μl for subsequent use.

[0052] In some embodiments, in step (3), a DNA origami crystal substrate loaded with gold nanoparticles at specific sites is prepared by a one-step method: The origami framework prepared in step (1) and the gold nanoparticles modified in step (2) are mixed at a molar ratio of 1:1.1. Subsequently, an excessive amount of poly-A sequence is added. After being evenly dispersed, it is placed in a crystal incubator and annealed twice within the temperature range of 50 - 20°C to obtain a DNA origami crystal substrate;

[0053] In some embodiments, in step (3), a DNA origami crystal substrate loaded with gold nanoparticles at specific sites is prepared by a one-step method: the origami framework prepared in step (1) and the gold nanoparticles modified in step (2) are mixed at a molar ratio of 1:1.0, and then an excessive amount of poly-A sequence is added. After being dispersed evenly, it is placed in a crystal incubator and annealed twice within a temperature range of 50 - 20 °C to obtain a DNA origami crystal substrate;

[0054] In step (4), the origami crystal substrate is washed to remove excess DNA segments and gold nanoparticles: the substrate obtained in step (3) is washed multiple times with 1XTAE and 12.5 mM Mg(CH3COO)2 solution;

[0055] In step (5), the order and timing of adding different displacement strands are regulated in the washed substrate, supplemented with constant-temperature heat treatment, to achieve the conversion of the crystal structure of the nanoparticle superlattice between multiple states: according to the pre-set crystal structure change route, the corresponding displacement strands are added to the washed substrate at an initial ratio of 1:5 for each inner strand and displacement strand. The displacement strands are divided into two types, R and P, where the R strand is called the pre-sealing strand when synthesizing various DNA origami framework structures in step (1); the nucleotide sequences of the displacement strands are as shown in SEQ NO: 17, 18, 19, 20, 21…24;

[0056] Subsequently, it is left standing at a constant temperature to prepare a structure-dynamically controllable nanoparticle superlattice based on a DNA origami crystal template. After adding the displacement strands, the origami crystal needs to be left standing at a constant temperature of 37 °C or 46 °C for 24 h. After the heat treatment is completed, the crystal needs to be washed with 1XTAE and 12.5 mM Mg(CH3COO)2 solution. The washed crystal is stored at 4 °C.

[0057] In some embodiments, a method for preparing a structure-dynamically controllable nanoparticle superlattice based on a DNA origami crystal template of the present invention includes the following steps:

[0058] In step (2), functionalized thiol DNA strands are modified on the surface of 10-nm gold nanoparticles: First, TCEP is used to reduce the DNA strands modified with disulfide bonds at one end to expose the thiol termini. Subsequently, purification is performed using a G-25 size exclusion centrifugal column. Then, the purified thiol DNA strands are mixed with 10-nm gold nanoparticles at a ratio of 300:1, and the reaction time at room temperature is 2 h. Subsequently, an appropriate amount of PB buffer is added to make its final concentration 10 mM. Then, 2 M NaCl solution is slowly added successively to make the final concentration of NaCl 0.3 M. The mixed solution is placed in a rotary reaction at room temperature for 24 h. Finally, the gold nanoparticles after the reaction are purified by high-speed centrifugation multiple times to remove the excess thiol DNA strands in the solution. The modified gold nanoparticles are dispersed in 0.1 M PBS buffer and stored statically at 4 °C;

[0059] In some embodiments, a method for preparing a structurally dynamically controllable nanoparticle superlattice based on a DNA origami crystal template of the present invention includes the following steps:

[0060] In step (2), functionalized thiol DNA strands are modified on the surface of 10-nm gold nanoparticles: First, TCEP is used to reduce the DNA strands modified with disulfide bonds at one end to expose the thiol termini. Subsequently, purification is performed using a G-25 size exclusion centrifugal column. Then, the purified thiol DNA strands are mixed with 10-nm gold nanoparticles at a ratio of 300:1, and the reaction time at room temperature is 1.8 h. Subsequently, an appropriate amount of PB buffer is added to make its final concentration 10 mM. Then, 2 M NaCl solution is slowly added successively to make the final concentration of NaCl 0.3 M. The mixed solution is placed in a rotary reaction at room temperature for 18 h. Finally, the gold nanoparticles after the reaction are purified by high-speed centrifugation multiple times to remove the excess thiol DNA strands in the solution. The modified gold nanoparticles are dispersed in 0.1 M PBS buffer and stored statically at 4 °C.

[0061] The second aspect of the present invention is to provide a structurally dynamically controllable nanoparticle superlattice prepared by a method for preparing a structurally dynamically controllable nanoparticle superlattice based on a DNA origami crystal template.

[0062] Example 1

[0063] A method for preparing a structurally dynamically controllable nanoparticle superlattice based on a DNA origami crystal template of the present invention includes the following steps:

[0064] This example starts from the initial state substrate and manipulates the sequential transformation of the nanoparticle crystal structure among six states, specifically including the following steps:

[0065] (1) Synthesis of four DNA origami framework structures: Mix the M13mp18 long chain, hundreds of staple strands, sticky-end strands, and inner strands at a molar ratio of 1:10:10:7.5 in a 1XTAE, 12.5 mM Mg(CH3COO)2 solution. The mixture undergoes a gradient cooling program from 90 - 20 °C in a PCR for 22 h, and finally DNA origami frameworks with a concentration of 10 nM are obtained. Among them, according to different origami shapes, three different sets of sequences are selected for the staple strands. The sticky-end strands and inner strands of the four monomers are different from each other and can thus be independently manipulated. Here, the monomers all have the ability to load nanoparticles in the initial state, so pre-capping is not required. The sequences of the staple strands and sticky-end strands can be found in the DNA sequence a, b, c, g in the supplementary information of the article Sci.Adv., 2022, 8, eadc9755, and the sequence information of the inner strands is shown in the sequence listing.

[0066] (2) Modify the 10 nm gold nanoparticles with functionalized thiol DNA strands. First, use TCEP to reduce the DNA strand modified with a disulfide bond at one end to expose the thiol terminus, and then purify it with a G-25 size exclusion centrifugal column. Next, mix the purified thiol DNA strand with 10 nm gold nanoparticles at a molar ratio of 300:1, react at room temperature for 1.5 h, then add the corresponding amount of PB buffer to make its final concentration 10 mM, and then slowly add 2 M NaCl solution successively to make the final concentration of NaCl 0.3 M. The mixed solution is placed at room temperature and rotated for reaction for 18 h. Finally, purify the reacted gold nanoparticles by high-speed centrifugation multiple times to remove the excess thiol DNA strands in the solution. The modified gold nanoparticles are dispersed in 0.1 M PBS buffer and stored at 4 °C.

[0067] (3) One-step preparation of the initial-state DNA origami crystal substrate: Mix the four DNA origami frameworks prepared in step (1) in equal proportions, then add the gold nanoparticles modified in step (2), and then add an excessive poly-A sequence at a molar ratio of each sticky-end strand: poly-A sequence of 1:10. The nucleotide sequence of the poly-A is as shown in SEQ NO: 25. After uniform dispersion, place it in a crystal incubator and anneal twice in the temperature range of 50 - 20 °C to obtain the initial-state DNA origami crystal substrate.

[0068] (4) Wash the initial-state substrate to remove the excess DNA segments and gold nanoparticles. Wash the substrate obtained in step (3) multiple times with a 1XTAE, 12.5 mM Mg(CH3COO)2 solution, and then volume-fix each tube of the substrate to 20 ul for subsequent use.

[0069] (5) Regulate the order and timing of adding different replacement chains in the washed substrate, supplemented by constant-temperature heat treatment, so as to realize the conversion of the crystal structure of the nanoparticle superlattice among multiple states. As Figure 1 shown, starting from the initial-state substrate, different types and proportions of replacement chains are added in different steps. If the R chain is added alone, the sample is placed at a constant temperature of 37 °C for 24 h. If the replacement chain contains the P chain, the sample is placed at a constant temperature of 46 °C for 24 h. After the heat treatment at each node is completed, the crystal is washed with a 1XTAE, 12.5 mM Mg(CH3COO)2 solution to remove the replacement chains in the system and fix the crystal phase in the solution at this time. A total of 6 times of replacement chains need to be added in this example, and the crystal structure of the nanoparticles goes through five states and finally returns to the initial state. The overall change process is characterized by small-angle X-ray scattering, as Figure 2 shown, and each curve represents the crystal structure of the nanoparticles at each transition node. The replacement chains are divided into two types, R and P, where the R chain is called the pre-sealing chain when synthesizing various DNA origami framework structures in step (1);

[0070] The nucleotide sequence of the inner chain is as shown in SEQ NO: 1, 2, 3, 4, 5…16;

[0071] The nucleotide sequence of the replacement chain is as shown in SEQ NO: 17, 18, 19, 20, 21…24;

[0072] The nucleotide sequence of poly-A is as shown in SEQ NO: 25.

[0073] Among them, the sequences 1-25 are shown in Table 1:

[0074]

[0075]

[0076] Example 2

[0077] The difference between Example 2 and Example 1 is that:

[0078] A preparation method of a structurally dynamically controllable nanoparticle superlattice based on a DNA origami crystal template of the present invention includes the following steps:

[0079] In this example, starting from the intermediate-state substrate, by manipulating the nanoparticles, its crystal structure is sequentially transformed into two other states.

[0080] In step (1), four DNA origami framework structures are synthesized: Mix the M13mp18 long chain, hundreds of staple strands, sticky-end strands, inner strands, and pre-sealing strands in a ratio of 1:10:10:7.5:30 in a 1XTAE, 12.5 mM Mg(CH3COO)2 solution. The mixture undergoes a gradient cooling program from 90 - 20 °C in a PCR for 23 h, and finally DNA origami frameworks with a concentration of 10 nM are obtained. Among them, according to different origami shapes, three different sets of sequences are selected for the staple strands. The sticky-end strands and inner strands of the four monomers are different from each other and can thus be independently manipulated. Two of the monomers involved here do not require the addition of pre-sealing strands and have the ability to load nanoparticles in the initial state, while the other two require the addition of pre-sealing strands so that they cannot capture nanoparticles in the initial state but can recover the ability to load nanoparticles in subsequent experiments.

[0081] In step (2), functionalized thiol DNA strands are modified on the surface of 10 nm gold nanoparticles. The specific experimental operation is the same as that in step (2) of Example 1.

[0082] In step (3), an intermediate-state DNA origami crystal substrate is prepared by a one-step method. Mix the four DNA origami frameworks prepared in step (1) in equal proportions, and then add the gold nanoparticles modified in step (2). Note that only the gold nanoparticles required for two monomers need to be added here. Then, an excessive amount of poly-A sequence is added at a molar ratio of each sticky-end strand:poly-A sequence of 1:10. After being dispersed evenly, it is placed in a crystal incubator and annealed twice in the temperature range of 50 - 20 °C to obtain an intermediate-state DNA origami crystal substrate.

[0083] In step (4), the intermediate-state substrate is washed. The specific experimental operation is the same as that in step (4) of Example 1.

[0084] In step (5), the order and timing of adding different displacement strands are regulated in the washed substrate, supplemented with constant-temperature heat treatment, so as to achieve the conversion of the nanoparticle superlattice among multiple states. As Figure 3 shown, starting from the intermediate-state substrate, different types and proportions of displacement strands are added in different steps. After the displacement strands R and P are added simultaneously, the sample is placed at 46 °C for constant-temperature treatment for 24 h. After the heat treatment at each node is completed, the crystal is washed with a 1XTAE, 12.5 mM Mg(CH3COO)2 solution to remove the displacement strands in the system and fix the crystal phase in the solution at this time. A total of 2 times of displacement strands need to be added in this example, and the crystal structure of the nanoparticles is sequentially transformed into two other states. As Figure 4 shown, the surface arrangements of the gold nanoparticles in the two obtained states are characterized by a scanning electron microscope.

[0085] Example 3

[0086] Example 3 is different from Example 1 in that: A method for preparing a structurally dynamically controllable nanoparticle superlattice based on a DNA origami crystal template of the present invention includes the following steps:

[0087] In step (1), four DNA origami framework structures are synthesized: Mix the M13mp18 long chain, hundreds of staple chains, sticky end chains, inner chains, and pre-sealed chains in a ratio of 1:10:10:7.5:30 in 1XTAE, 12.5 mM Mg(CH3COO)2 solution. The mixture undergoes a gradient cooling program with a temperature range of 90 - 20 °C in a PCR for 24 hours, and finally DNA origami frameworks with a concentration of 10 nM are obtained.

[0088] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection required by the present invention is defined by the appended claims, the specification and their equivalents.

Claims

1. A method for preparing a structure-dynamically controllable nanoparticle superlattice based on a DNA origami crystal template, characterized in that The steps include: (1) Synthesis of various DNA origami framework structures: The DNA origami is composed of M13mp18 long chains, staple chains, sticky end chains, inner chains and pre-blocking chains; the M13mp18 long chains, hundreds of staple chains, sticky end chains, inner chains and pre-blocking chains are mixed in a molar ratio of 1:10:10:7.5:30 in 1XTAE, 12.5 mM Mg(CH3COO)2 solution, and the mixture is subjected to a gradient cooling program from 90 to 20 °C in PCR for 22 to 24 h, and finally a DNA origami framework with a concentration of 10 nM is obtained; the nucleotide sequence of the inner chain is shown in SEQ ID NO: 1-16; (2) Modification of functionalized thiol DNA chains on the surface of 10 nm gold nanoparticles: First, TCEP was used to reduce the DNA chain modified with a disulfide bond at one end to expose the thiol end, and then it was purified using a G-25 size exclusion centrifugal column. Next, the purified thiol DNA chain was mixed with 10 nm gold nanoparticles at a ratio of 300:1, and the reaction time was 1.5-2 h at room temperature. Then, the corresponding amount of PB buffer was added to make the final concentration of 10 mM, and then 2M NaCl solution was slowly added gradually to make the final concentration of NaCl 0.3 M. The mixed solution was placed at room temperature for rotation reaction for 18-24 h. Finally, the gold nanoparticles after the reaction were purified by multiple high-speed centrifugation to remove the excess thiol DNA chain in the solution. The modified gold nanoparticles were dispersed in 0.1 M PBS buffer and stored at 4 °C. (3) One-step preparation of a DNA origami crystal substrate loaded with gold nanoparticles at specific sites: the origami framework prepared in step (1) and the gold nanoparticles modified in step (2) are mixed at a molar ratio of 1:0.9-1:1, and then an excess of poly-A sequence is added. After being evenly dispersed, the mixture is placed in a crystal incubator and annealed twice in a temperature range of 50-20°C to obtain a DNA origami crystal substrate; the nucleotide sequence of the poly-A is as shown in SEQ ID NO: 25; the poly-A sequence is added in excess at a molar ratio of each sticky end chain: poly-A sequence of 1:10; (4) Washing the origami crystal substrate to remove excess DNA segments and gold nanoparticles: Wash the substrate obtained in step (3) multiple times using 1XTAE, 12.5 mMM Mg(CH3COO)2 solution; (5) The order and timing of adding different displacement chains to the washed substrate are regulated, and constant temperature heat treatment is performed to realize the crystal structure conversion of the nanoparticle superlattice between multiple states: according to the pre-set crystal structure change route, the corresponding displacement chains are added to the washed substrate at an initial molar ratio of each inner chain to the displacement chain of 1:

5. The displacement chains are divided into two types, R and P. The nucleotide sequence of the R chain is shown in SEQ ID NO: 17-20, and the nucleotide sequence of the P chain is shown in SEQ ID NO: 21-24. Among them, the R chain is called the pre-blocking chain when synthesizing multiple DNA origami framework structures in step (1); then the sample is kept at a constant temperature. If the R chain is added alone, the sample is kept at a constant temperature of 37°C for 24 hours. If the P chain is included in the displacement chain, the sample is kept at a constant temperature of 46°C for 24 hours to obtain a structurally dynamically controllable nanoparticle superlattice based on a DNA origami crystal template.

2. The method for preparing a structure-dynamically controllable nanoparticle superlattice based on a DNA origami crystal template according to claim 1, characterized in that: In step (1), the staple chain, sticky end chain, inner chain, and pre-sealing chain should be appropriately selected according to the shape, connection capacity, and load capacity of the origami frame, involving a total of eight different origami monomers.

3. The method for preparing a structure-dynamically controllable nanoparticle superlattice based on a DNA origami crystal template according to claim 1, characterized in that: In step (3), the origami crystal substrate is divided into two categories: initial state and intermediate state. Regardless of which category, four different DNA origami frameworks are required. All monomers of the initial state substrate can load nanoparticles, while the intermediate state substrate can only load nanoparticles at specific sites. The amount of gold nanoparticles added is calculated based on the origami monomers with loading capacity.

4. The method for preparing a structure-dynamically controllable nanoparticle superlattice based on a DNA origami crystal template according to claim 1, characterized in that: In step (4), after washing the substrate, each tube of substrate needs to be adjusted to 20 ul for subsequent use.

5. The method for preparing a structure-dynamically controllable nanoparticle superlattice based on a DNA origami crystal template according to claim 1, characterized in that: In step (5), after the heat treatment, the crystals are washed with 1XTAE, 12.5 mM Mg(CH3COO)2 solution; the washed crystals are stored at 4°C.

6. A nanoparticle superlattice with dynamically controllable crystal structure obtained by the method for preparing a nanoparticle superlattice with dynamically controllable structure based on a DNA origami crystal template as described in any one of claims 1 to 5.

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