CN115011595A - DNA Origami nano particle with any size and preparation - Google Patents
DNA Origami nano particle with any size and preparation Download PDFInfo
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- CN115011595A CN115011595A CN202210855667.4A CN202210855667A CN115011595A CN 115011595 A CN115011595 A CN 115011595A CN 202210855667 A CN202210855667 A CN 202210855667A CN 115011595 A CN115011595 A CN 115011595A
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- 239000002105 nanoparticle Substances 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 14
- 108020004414 DNA Proteins 0.000 claims description 60
- 238000002156 mixing Methods 0.000 claims description 12
- 101710163270 Nuclease Proteins 0.000 claims description 6
- 238000001976 enzyme digestion Methods 0.000 claims description 6
- 102000053602 DNA Human genes 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 2
- 239000002086 nanomaterial Substances 0.000 description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000008049 TAE buffer Substances 0.000 description 3
- HGEVZDLYZYVYHD-UHFFFAOYSA-N acetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound CC(O)=O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O HGEVZDLYZYVYHD-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 3
- 108010086093 Mung Bean Nuclease Proteins 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
Abstract
DNA Origami nano particles with any size are prepared, and the size of the DNA Origami nano particles is smaller than the size determined by a scaffold chain or is a non-integral multiple of the size appointed by the scaffold chain; the preparation method comprises the following steps: designing rectangular DNA Origami, regarding the rectangular DNA Origami as canvas, and regarding each staple chain as an individual pixel; selecting a staple chain corresponding to the corresponding pixel according to the required structure; the scaffold chain and the selected staple chain are mixed together for assembly; the remaining single strand on the scaffold chain is then removed. The method can effectively reduce the design pressure, break through the limitation of size, improve the utilization rate of the staple chain, reduce the time and the economic cost, further expand the freedom degree of the technology of DNA Origami and realize the low-cost assembly of the DNA Origami with different sizes and controllable sizes.
Description
Technical Field
The invention relates to the field of DNA self-assembly, in particular to DNA Origami nano particles with any size and preparation thereof.
Background
DNA Origami is a novel DNA nanostructure assembly technique, first proposed in 2006 by professor Rothemund of the California institute of science and technology (Scafffolded DNA Origami for nanoscales and patterns [ J ]. Journal of the American Chemical Society,2006,231), which has greatly facilitated the development and application of DNA nanotechnology. This method is mainly to fold a phage DNA having a length of about 7000 bases into a previously designed shape by the principle of base complementary pairing using two hundred short oligonucleotide strands called "staple strands" and phage DNA (M13mp18) called "scaffold strands". The DNA Origami assembly process specifically comprises the following steps: (1) determining a target graph; (2) folding the scaffold chain according to the target graph to enable the shape formed by folding the scaffold chain to meet the requirement of the target graph; (3) designing sequences of staple chains according to the folding structures of the scaffold chains, each staple chain being complementary to a reverse sequence adjacent to the folding structures of the scaffold chains, so that a cross structure is formed to connect adjacent sequences of the scaffold chains together; (4) and mixing the scaffold chain and the designed staple chain in proportion, and then annealing and assembling. The DNA Origami has great freedom, can be designed and assembled to obtain a two-dimensional or three-dimensional nano structure in any shape, and can also be used as a unit structure to be assembled to obtain a larger structure, for example, two dolphin-shaped DNA Origami are connected together, or 9 small square DNA Origami are combined to form a larger rectangular structure.
The DNA Origami is not only a nanostructure formed by folding a scaffold chain alone, but also a solid pattern formed by filling a double-helix structure formed by the scaffold chain and a staple chain. The DNA Origami structure contains a plurality of DNA cross structures, so that the DNA Origami nano structure can stably exist in a conventional environment. The position of each staple chain and each base in the DNA nanostructure can be precisely positioned, so that the DNA Origami has excellent addressing performance. The requirement on the stoichiometric ratio of the staple chain is not high in the assembly process of the DNA Origami, and the assembly process is simple. Compared with other nanoparticle synthesis methods, the DNA Origami can obtain nanoparticles with any shape only by mixing scaffold chains and staple chains and then annealing, and has very high degree of freedom and accuracy. The assembled DNA Origami nanoparticles can also be connected together through sticky ends to obtain a larger structure.
Although the DNA Origami has the advantages of large degree of freedom, precise shape, simple assembly and the like, the DNA Origami also has certain defects. Particularly, as the demand is higher, a series of problems such as the limitation of the length of the scaffold chain, the complexity of the design of the staple chain, the repeated utilization rate of the staple chain with different patterns, and the like, on the size of the DNA Origami are gradually exposed. First, the utilization of staple chains is low, and a set of staple chains containing more than two hundred short chains can only be used to assemble one nanostructure. Different designs require different staple chains and two hundred staple chains must be redesigned and combined, which results in increased labor and time and economic costs as the design increases. Second, the scaffold strands used by DNA Origami are of fixed length, so that the surface area of the assembled nanostructure is nearly identical. Because multiple assembled nanostructures can be connected by sticky ends, the total surface area of the designed and assembled nanostructures is an integer multiple of the surface area of a single nanostructure. The size of the nanostructure is greatly limited by the conventional DNA Origami, and the size of the nanostructure cannot be smaller than that of the nanostructure obtained by folding one scaffold chain, and must be integral multiple of the size.
Disclosure of Invention
The present invention is directed to solving the above problems in the prior art, and provides DNA Origami nanoparticles of any size and preparation thereof, which can effectively reduce the design pressure, break through the limitation of size, improve the utilization rate of staple chains, reduce time and economic cost, further expand the degree of freedom of DNA Origami technology, and realize low-cost assembly of DNA Origami with different sizes and controllable sizes.
In order to achieve the purpose, the invention adopts the following technical scheme:
DNA Origami nanoparticles of any size, the size of the DNA Origami nanoparticles being smaller than the size determined by the scaffold strand, or the size of the DNA Origami nanoparticles being a non-integer multiple of the size agreed upon by the scaffold strand.
The preparation of the DNA Origami nano-particles with any size comprises the following steps:
1) designing rectangular DNA Origami, regarding the rectangular DNA Origami as canvas, and regarding each staple chain as an individual pixel;
2) selecting a staple chain corresponding to the corresponding pixel according to the required structure; mixing the scaffold chain and the selected staple chain together for assembling; the remaining single strand on the scaffold chain is then removed.
In the step 1), the canvas consists of a scaffold chain and a plurality of staple chains, and the scaffold chain and the staple chains are assembled to obtain rectangular DNA Origami.
In the step 2), the structure obtained according to the requirement is formed by pixels in a canvas, corresponding staple chains are selected according to the pixels, and sequences corresponding to the staple chains and the scaffold chains form double chains in the assembling process to form pixels on the rectangular canvas; the sequence corresponding to the scaffold chain corresponding to the unselected staple chain remains single-stranded because there is no corresponding scaffold chain to form a double strand with it.
In the step 2), removing the residual single chains on the scaffold chain by adopting an enzyme digestion method.
The enzyme digestion method is to use nuclease to carry out enzymolysis on the sequence which still keeps the single-stranded state after assembly so as to remove the sequence, and the double-stranded part still keeps the assembled state.
The nuclease is adopted as the enzyme, and can only degrade single-stranded DNA fragments without influencing double-stranded DNA fragments.
In the invention, a plurality of DNA Origami nanoparticles obtained by assembly can be connected through sticky ends, and the size of the obtained nanoparticles is larger than the size appointed by a scaffold chain and is not integral multiple of the size.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the invention designs a rectangular DNA Origami and obtains the position and sequence information of all staple chains; regarding the rectangular DNA Origami as a canvas, regarding each staple chain as an individual pixel; selecting a staple chain corresponding to the corresponding pixel according to a desired structure; the scaffold chain and the selected staple chain are mixed together for assembly; the remaining single strands of the scaffold strands are specifically digested with nucleases to give a desired structure consisting entirely of double strands. The method can realize the assembly of various nano structures with different sizes, namely the size of the nano structure can be smaller than that determined by the length of a scaffold chain; multiple nanostructures are joined by sticky ends, allowing for structures in which individual nanostructures are not integer multiples of the size. A set of staple chains can be assembled in any shape and size configuration, eliminating the need to redesign and synthesize staple chains from different designs, reducing design stress and reducing time and cost.
Drawings
FIG. 1 is a schematic diagram of a rectangular canvas;
FIG. 2 is a schematic diagram of a rectangular canvas composition;
FIG. 3 is a schematic view of a triangular nanoparticle;
FIG. 4 is a schematic view of an "E" shaped hollow nanoparticle.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.
Example 1
Fig. 1 is a synthesized rectangular canvas, and a schematic diagram of a synthesis process of the synthesized rectangular canvas is shown in fig. 2. Mixing (1) long chain M13mp18 and (2) rectangular 216 staple chains at a concentration ratio of 1: 5 in a mixture containing 12.5mM Mg 2+ 1 XTAE (Tris-acetate acid,0.02 mM; EDTA,2mM) buffer; mixing, placing into PCR instrument, and slowly cooling from 95 deg.C to 15 deg.C at a rate of 1 deg.C per 2 min; taking out, and ultrafiltering with 1 × TAE buffer solution for 5 times to obtain (3) rectangular canvas.
Example 2
Figure 3 is an assembled triangle that is smaller than the size of the canvas. Selecting corresponding triangular staple chain according to rectangular canvas, mixing long chain M13mp18 and selected staple chain at concentration ratio of 1: 5 in the mixture containing 12.5mM Mg 2+ 1 XTAE (Tris-acetate acid,0.02 mM; EDTA,2mM) buffer;mixing, placing into PCR instrument, and slowly cooling from 95 deg.C to 15 deg.C at a rate of 1 deg.C per 2 min; taking out, and ultrafiltering with 1 × TAE buffer solution for 5 times to remove excessive short chain; washing the purified DNA origami structure once by using a new ultrafiltration tube with an acetic acid buffer solution with pH 5.2; mixing the obtained sample with mung bean nuclease to form a 20 mu l reaction system, carrying out enzyme digestion at 37 ℃ for 15min, and rapidly adding 2 mu l 2mM EDTA to terminate the reaction, thereby obtaining the triangular nanoparticles.
Example 3
Fig. 4 shows an assembled hollow shape "E". Selecting staple chain according to the requirement of corresponding pattern by matching with rectangular canvas, mixing long chain M13mp18 and selected staple chain at a concentration ratio of 1: 5 in a mixture containing 12.5mM Mg 2+ 1 XTAE (Tris-acetate acid,0.02 mM; EDTA,2mM) buffer; mixing, placing into PCR instrument, and slowly cooling from 95 deg.C to 15 deg.C at a rate of 1 deg.C per 2 min; taking out, and ultrafiltering with 1 × TAE buffer solution for 5 times to remove excessive short chain; washing the purified DNA origami structure once by using a new ultrafiltration tube with an acetic acid buffer solution with the pH of 5.2 to obtain a double-chain and loose single-chain combination; mixing the obtained sample with mung bean nuclease to form a 20 mu l reaction system, carrying out enzyme digestion at 37 ℃ for 15min, and rapidly adding 2 mu l of 2mM EDTA to terminate the reaction, thereby obtaining the hollow-out-shaped 'E' nanoparticles.
Claims (8)
1. DNA Origami nanoparticles of any size characterized by: the size of the DNA Origami nano-particle is smaller than the size determined by the scaffold chain, or the size of the DNA Origami nano-particle is non-integral multiple of the size appointed by the scaffold chain.
2. The preparation of DNA Origami nanoparticles of any size according to claim 1, characterized in that it comprises the following steps:
1) designing rectangular DNA Origami, regarding the rectangular DNA Origami as canvas, and regarding each staple chain as an individual pixel;
2) selecting a staple chain corresponding to the corresponding pixel according to the required structure; mixing the scaffold chain and the selected staple chain together for assembling; the remaining single strand on the scaffold chain is then removed.
3. Preparation of DNA Origami nanoparticles of any size according to claim 2, characterized in that: in the step 1), the canvas consists of a scaffold chain and a plurality of staple chains, and the scaffold chain and the staple chains are assembled to obtain rectangular DNA Origami.
4. Preparation of DNA Origami nanoparticles of any size according to claim 2, characterized in that: in the step 2), the structure obtained according to the requirement is formed by pixels in a canvas, corresponding staple chains are selected according to the pixels, and sequences corresponding to the staple chains and the scaffold chains form double chains in the assembling process to form pixels on the rectangular canvas; the sequence corresponding to the scaffold chain corresponding to the unselected staple chain remains single-stranded because there is no corresponding scaffold chain to make up a double strand with it.
5. Preparation of DNA Origami nanoparticles of any size according to claim 2, characterized in that: in the step 2), removing the residual single chains on the scaffold chain by adopting an enzyme digestion method.
6. Preparation of DNA Origami nanoparticles of any size according to claim 5, characterized in that: the enzyme digestion method is to use nuclease to carry out enzymolysis on the sequence which still keeps the single-stranded state after assembly so as to remove the sequence, and the double-stranded part still keeps the assembled state.
7. Preparation of DNA Origami nanoparticles of any size according to claim 6, characterized in that: the nuclease is adopted, and can only degrade single-stranded DNA fragments, so that the nuclease has no influence on double-stranded DNA fragments.
8. Preparation of DNA Origami nanoparticles of any size according to claim 2, characterized in that: the assembled plurality of DNA Origami nano-particles can be connected through sticky ends, and the size of the nano-particles is larger than the size appointed by the scaffold chain and is not integral multiple of the size.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210855667.4A CN115011595A (en) | 2022-07-18 | 2022-07-18 | DNA Origami nano particle with any size and preparation |
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| CN202210855667.4A CN115011595A (en) | 2022-07-18 | 2022-07-18 | DNA Origami nano particle with any size and preparation |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1692157A (en) * | 2002-03-07 | 2005-11-02 | 株式会社产学连携机构九州 | DNA nanocage by self-organization of DNA and process for producing the same, and DNA nanotube and molecule carrier using the same |
| CN102559660A (en) * | 2012-01-17 | 2012-07-11 | 生工生物工程(上海)有限公司 | Method for preparing double-stranded deoxyribonucleic acid (DNA) molecule with protruding end |
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- 2022-07-18 CN CN202210855667.4A patent/CN115011595A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1692157A (en) * | 2002-03-07 | 2005-11-02 | 株式会社产学连携机构九州 | DNA nanocage by self-organization of DNA and process for producing the same, and DNA nanotube and molecule carrier using the same |
| CN102559660A (en) * | 2012-01-17 | 2012-07-11 | 生工生物工程(上海)有限公司 | Method for preparing double-stranded deoxyribonucleic acid (DNA) molecule with protruding end |
Non-Patent Citations (2)
| Title |
|---|
| XIAOXING CHEN ET AL.: "Aptamer-Integrated Scaffolds for Biologically Functional DNA Origami Structures", ACS, vol. 13, pages 39711 - 39718 * |
| 王静文等: "酶辅助构建DNA纳米结构及其在无机纳米材料合成中的应用研究", 中国优秀硕士学位论文全文数据库工程科技I辑, pages 27 * |
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