CN114438079B - Virus-like DNA polyhedral framework structure and preparation method and application thereof - Google Patents
Virus-like DNA polyhedral framework structure and preparation method and application thereof Download PDFInfo
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- 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
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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Abstract
The invention discloses a virus-like DNA polyhedron frame structure, which is a regular icosahedron frame structure, each side consists of 4DNA double-spirals, and the structure is provided with an outer layer structure and an inner layer structure, wherein each side at the top of the outer layer structure is bridged by 5T single-stranded DNA, and each side at the top of the inner layer structure is bridged by 1T. In addition, the invention also relates to a packaging assembly composition containing the DNA material and a construction and manufacturing method of the composition. Finally, the invention also relates to the application of the DNA polyhedral framework structure in the fields of nanometer space molecular operation level, cell-free in-vitro amplification system and virus infection imitation. Compared with the prior art, the DNA polyhedron framework structure disclosed by the invention has a unique double-layer structure and more anchor points, so that the structure rigidity is better, the assembly prepared from the DNA polyhedron framework structure also has better integrity, the package is more complete, and the application of the DNA polyhedron framework structure in various fields is substantially improved compared with the prior art.
Description
Technical Field
The invention relates to the technical field of DNA (deoxyribonucleic acid) nanometer, in particular to a DNA polyhedral framework structure with a regular icosahedron structure, and a preparation method and application thereof.
Background
Nanomaterial refers to a material having at least one dimension in three dimensions in a nanometer size (1-100 nm) or consisting of them as a basic unit. Among numerous nanomaterials and related technologies, nucleic acid nanotechnology has demonstrated great advantages in terms of its precise structural editing capabilities, spatial organization capabilities and flexible and reliable controllable change capabilities for various other materials since the invention of DNA origami in 2006. The DNA molecule is a molecule which is ubiquitous in the nature, is not only a carrier for storing and transmitting genetic information of organisms, but also is a nano construction material with great potential due to the predictability and programmability of complementary pairing, can be bound and precisely folded to form a specific two-dimensional and three-dimensional structure through a short chain, and can be used as a natural nano material to participate in various functional structures and the construction of nano devices.
The artificial construction of nanostructures by DNA self-assembly has mainly two ideas: (1) if the graph is regarded as a house, we can find building materials, pile the building materials one by one brick and one tile, namely tile self-assembly; (2) if the graphic is to be considered a piece of clothing, it can be sewn out in a line with a set of stitches, i.e. DNA origami. The basic principle of DNA origami is to use several or even hundreds of Single-strand DNA (ssDNA) to recognize each other and locally pair them to form a double strand, and finally to weave the whole into a specific design structure. It has a number of distinct advantages such as design controllability, modifiable, low toxicity, low immunogenicity, etc. Using DNA nanotechnology, one can construct various nanoscale shapes from one-dimensional to three-dimensional, straight to curved, such as: tubular, annular, polyhedral, football, vase, etc.
Because of the precise addressability of the surface of the DNA nanostructure, it can also serve as a template to further guide the precise assembly (control of number, position, distance) of other nanomaterials, forming composite nanomaterials and implementing their application as tools, for example, DNA nanorings developed by applicant in 2016 guide the work of assembling phospholipid molecules to form artificial lipids with controllable size and shape.
In addition, framework structures based on DNA origami can also be used in viroid studies. Viruses are a typical biological macromolecule assembly, have a three-dimensional structure with a nanometer size and accuracy, perform definite biological functions, are a natural example of synthesizing nanometer materials through molecular self-assembly, and attract the attention of students and developers in more and more different fields. The basic structure of a virus consists of a protein shell (i.e., capsid) and genomic nucleic acid encapsulated therein. Viral capsids can be classified into spheres, rods, filaments, tadpoles, etc. according to morphology, spherical viral capsids often have regular icosahedral symmetry. Many viral capsid proteins can undergo reversible self-assembly to form structures similar to viral capsids, which can be referred to as viral nanoparticles, as components of building composite nanostructures and materials. The scale range of the DNA self-assembled nano structure is very consistent with the size range of viruses, the intersection of virology and nano technology generates an emerging field of virus nano technology, and the spherical virus imitation structural design such as regular octahedron and regular icosahedron is reported in the literature at present.
In recent years, with the development of nano materials and nano technology and the continuous deep cognition of human beings on life systems and life processes, the fusion and cross research of the nano technology and the biological medicine field is receiving a great deal of attention. At present, nucleic acid nano technology is continuously striving to provide a new method for solving medical problems, and the nucleic acid nano material has excellent application value in various aspects such as the biomedical field, wherein part of products are in clinical application, and a large number of products are in clinical trial stage. However, few nano materials with icosahedron structures exist in the prior art, and few nano materials with icosahedron structures have single-layer structures and have low rigidity; in addition, although there are reports of the structural design of a pseudo-spherical virus, there is no study of the packaging process and downstream functions of the pseudo-viral genome using a nucleic acid nanostructure. Therefore, it is very interesting to study a more rigid nucleic acid nanomaterial and to study the packaging process and downstream functions of the simulated viral genome using nucleic acid nanotechnology.
Disclosure of Invention
In order to solve at least one of the above problems, the present invention provides a novel DNA polyhedral frame structure, which has higher rigidity and better package integrity than the existing DNA polyhedral materials.
The above-mentioned DNA polyhedron frame structure is a regular icosahedron frame structure, each side is composed of 4DNA double helices (4 x Helix side structure), and has:
an outer layer structure, wherein the edges at the vertex are bridged by 5T single-stranded DNA (5T-spacer);
in the inner layer structure, each edge at the vertex is bridged by 1T (1T-spacer).
In the above structure, the regular icosahedron framework structure comprises 30 sides, and each side can be extended inwards or outwards to form an anchor point sequence for grabbing a genome or functionalizing the structure.
The invention also relates to a method for constructing the nano material.
The method comprises the following steps of: constructing a regular icosahedron framework structure, and presenting the structural schematic diagram by utilizing a 2D/3D image;
step 2: further constructing the edge and the inner and outer layer structures of the regular icosahedron on the basis of the step 1;
step 3: further constructing inner and outer modifiable sites of the regular icosahedron DNA framework structure on the basis of the step 2;
step 4: and (3) carrying out structural assembly and purification on the regular icosahedron DNA framework structure constructed in the step (3).
In the method, the step 1 subsequently comprises the steps of obtaining corresponding sequences of hundreds of staple chains through inputting template chain sequences, and synthesizing the sequences; the step 4 comprises removing unbound single strands of the residual materials and impurities such as dimers and multimers by using an ultracentrifugation and ultrafiltration concentration method.
The invention also relates to a packaging assembly composition related to the DNA polyhedron framework structure, and an regular icosahedron DNA framework structure with an anchoring chain; and
single-or double-stranded circular DNA capable of base complementary pairing with the anchor strand of the regular icosahedron DNA framework structure.
In the above composition, the single-stranded circular DNA is selected from phiX174 genome comprising 5386 bases, and 30 segments of 20 or more base sequences are selected as anchor fragments.
The invention also relates to a method for preparing the composition, which comprises the following steps:
step 1: encoding the 30 sides of the imitated viral DNA polyhedral frame structure according to claim 2 in an end-to-end sequence, wherein each side extends out of an anchor chain with the length of 20 bases or more;
step 2: selecting 30 sections of 20 or more base sequences on a single-stranded circular DNA containing 5386 bases as anchor fragments, and performing base complementary pairing with an anchor strand extending out of an icosahedron;
step 3: the packaging composition is purified to remove unbound single strands, as well as some dimers, multimers, etc. from the system.
In addition, the invention also relates to the application of the DNA polyhedral frame structure in the nano space molecular operation level, the application in a cell-free in-vitro amplification system and the application in a virus infection imitating link.
The invention has the advantages and beneficial effects that:
(1) the invention provides a DNA polyhedral frame structure and a preparation method thereof, wherein the material is a regular icosahedron and has a unique double-layer structure, and compared with the existing material, the material has better structural rigidity and is not easy to collapse;
(2) compared with the existing materials, the DNA polyhedral frame structure has more anchor points, so that the assembly prepared by the DNA polyhedral frame structure has better integrity and more complete package;
(3) through experiments, the DNA polyhedral framework structure and the assembly with the DNA polyhedral framework structure have good application capability in the nano space molecular operation level (especially single-chain DNA molecular biological operation), a cell-free in-vitro amplification system and the field of virus infection imitation, and have substantial and obvious progress compared with the prior material.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following description will briefly introduce the drawings that are required to be used in the embodiments or the prior art descriptions, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a prior art DNA polyhedral framework and its packaged assembly composition;
FIG. 2 is a schematic diagram showing the structure of a DNA polyhedral frame structure and a packaging assembly composition thereof in an embodiment of the present invention;
FIG. 3 is a graph comparing the packaging efficiency of a packaged fitment composition in one embodiment of the invention with other compositions;
FIG. 4 is a diagram showing the results of verification of the ability of a DNA polyhedral framework and its packaging assembly composition to manipulate a molecule in a confined space in accordance with one embodiment of the present invention;
FIG. 5 is a diagram showing the results of verification of the ability of a DNA polyhedral framework and its packaging assembly composition to manipulate a molecule in a confined space in accordance with another embodiment of the present invention;
FIG. 6 is a graph of the results of an in-structure rolling circle replication experiment of a packaged assembly composition in one embodiment of the invention;
FIG. 7 is a graph showing the verification result of the virus infection imitating ability of the DNA polyhedral framework structure and the packaging assembly composition in the embodiment of the invention.
FIG. 8 is a schematic diagram of the structure of a polyhedral frame structure of a virus-like DNA according to an embodiment of the present invention.
Detailed Description
The following describes the embodiments of the present invention further with reference to the drawings and examples. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
The invention relates to a regular icosahedron DNA polyhedral framework structure with a double-layer structure and a packaging assembly composition thereof under the background of DNA paper folding, and also provides a method for manufacturing the DNA polyhedral framework structure and the packaging assembly composition thereof. In a further aspect, the present invention also relates to the use of the above-mentioned materials and their compositions at different levels, for reference by a person skilled in the art.
Virus-like DNA polyhedral frame structure
As shown in FIG. 8, the present invention describes a DNA polyhedral frame structure, which is a regular icosahedron frame structure, each side of which is composed of 4DNA double helices (4 x Helix side structure), the 4x Helix side structure can make the regular icosahedron structure more rigid, preferably, a regular icosahedron frame structure comprises 30 sides, each side of which can extend inward or outward for grasping the genome downstream. In addition, the regular icosahedron framework structure also has an outer layer structure and an inner layer structure, wherein each side at the vertex of the outer layer structure is bridged by 5T single-stranded DNA (5T-spacer), and each side at the vertex of the inner layer structure is bridged by 1T (1T-spacer). The unique regular icosahedron and double-layer structure can make the DNA polyhedral frame structure have stronger rigidity.
Fig. 1 is a schematic structural view of a DNA polyhedral frame structure and a packaging assembly composition thereof related to the prior art, and from the view of an imaging chart, the DNA polyhedral frame structure and the packaging assembly composition thereof are poor in rigidity and easy to collapse, and fig. 2 is a schematic structural view of the DNA polyhedral frame structure and the packaging assembly composition thereof in the embodiment of the invention, and compared with the DNA polyhedral frame structure and the packaging assembly composition thereof shown in fig. 1, the DNA polyhedral frame structure and the packaging assembly composition thereof have uniform shape, clear edges and corners, do not collapse and have very strong rigidity under an electron microscope.
Construction of DNA polyhedral framework
The invention also discloses a method for constructing the DNA polyhedral framework structure, which specifically comprises the following steps:
step 1: and constructing a regular icosahedron framework structure. This step may utilize 2D or 3D image rendering techniques to design the DNA polyhedral framework structure to which the present invention relates, preferably the two-layer icosahedral structure map may be designed by Tiamat and cadnan; as a preferred embodiment, step 1 further comprises the following steps: preparing a template chain sequence, obtaining corresponding sequences of hundreds of staple chains through inputting the template chain sequence, and performing sequence synthesis after the completion.
Step 2: after the frame structure of the regular icosahedron is constructed, the edge and the inner and outer layer structures of the regular icosahedron are further constructed;
step 3: after the construction of the edge and the inner and outer layer structures of the regular icosahedron is completed, further constructing the inner and outer layer modifiable sites of the regular icosahedron DNA polyhedral framework structure;
step 4: after the construction of the inner and outer modifiable sites is completed, the structural assembly and purification of the regular icosahedron DNA polyhedral framework structure are carried out to remove the remaining unbound single strands and some dimers, multimers and the like, and preferably, the purification modes mainly comprise an ultracentrifugation method, an ultrafiltration concentration method and the like.
After the synthesis of the positive icosahedron, the synthesis of the icosahedron can be verified by the characterization methods such as gel electrophoresis, negative electron microscope, frozen electron microscope and the like.
The ultracentrifugation method is a method of separating a polymer using an ultracentrifuge. When a solution containing a polymer (e.g., a solution of a nucleic acid and a protein) is subjected to a strong centrifugal force, the concentration of the polymer is higher than that of the solution, and the separation is achieved. The rate of sedimentation is related to both the size and density of its molecules and the molecular density and viscosity of the solution and the shape of its molecules. The most common are the sedimentation velocity method and the sedimentation equilibrium method.
The ultrafiltration is a membrane separation method for realizing selective separation and recovery of substances by utilizing the micropore structure of a semipermeable membrane and taking certain external pressure (0.1-0.5 MPa) as driving force. When the solution flows through the surface of the ultrafiltration membrane with a certain aperture at a certain flow rate, under the action of external pressure, solute and water with molecular weight smaller than the molecular weight of the membrane interception molecular weight in the solution permeate through the ultrafiltration membrane to form filtrate (abbreviated as filtrate), and solute with molecular weight larger than the molecular weight of the membrane interception molecular weight is intercepted by the membrane. The solution is gradually concentrated along with the progress of ultrafiltration, and when the solution reaches a certain concentration degree, the solution is discharged in the form of concentrated solution (also called mother solution), and solutes with different molecular weights in the solution are separated or concentrated.
Agarose or polyacrylamide gel electrophoresis is one of the nuclear techniques of gene manipulation, which can be used to isolate, identify and purify DNA fragments. The technology is simple and rapid to operate, and has become the technical foundation of a plurality of general molecular biology research methods, such as DNA recombination, DNA nucleotide sequence analysis, DNA restriction enzyme analysis, restriction enzyme mapping and the like.
The negative staining technique is a technique for preparing images of electron microscope samples to exhibit complex contrast. For observing particulate matter or biological macromolecules in a sample.
The cryoelectron microscope is an ultralow temperature freezing sample preparation and transmission technology (Cryo-TEM) for a transmission electron microscope, and can directly observe liquid, semi-liquid and samples sensitive to electron beams.
Packaging assembly groupComposition
The invention also describes a packaging assembly composition of a DNA polyhedral framework structure, which comprises a regular icosahedral DNA polyhedral framework structure with an anchor chain and a single-stranded circular DNA capable of carrying out base complementary pairing with the anchor chain of the regular icosahedral DNA polyhedral framework structure, wherein the single-stranded circular DNA is selected from phiX174 genome containing 5386 bases, and 30 segments of 20 base sequences or more are selected as anchor fragments.
phiX174, a phage of E.coli phiX174, is in the form of: icosahedral capsids have 12 spike-like projections, no tails, and a capsid size of 23 nm, a DNA virus that was earlier found to have a single-stranded, circular genome. The DNA strand packaged into the virion is called the "+" strand. After entry into the cell, phiX174DNA serves as a template for negative strand synthesis, producing double stranded DNA. Conversion of the +dna strand to double stranded DNA does not require any phage genes to function.
Construction of packaging Assembly composition
The invention discloses a method for constructing a packaging composition containing a single-chain circular phage genome, which specifically comprises the following steps:
step 1: the 30 edges contained in the regular icosahedron are encoded sequentially. The step encodes 30 sides of the DNA polyhedron frame structure in an end-to-end sequence, each side extends out of an anchor chain with 20 or more base lengths, and the encoding mode is preferably one stroke;
step 2: the phiX174 genomic single-stranded circular DNA anchor fragment was selected. Selecting 30 sections of 20 or more base sequences on a single-stranded circular DNA containing 5386 bases as anchor fragments, and performing base complementary pairing with an anchor strand extending out of an icosahedron;
step 3: preparation and purification of structures containing complementary anchor strands. After successful packaging, the packaged composition is purified to remove unbound single strands, as well as some dimers, multimers, etc., remaining in the system, preferably by ultracentrifugation and ultrafiltration concentration.
The DNA polyhedral frame structure constructed by the method and the packaging assembly composition thereof have good rigidity and stability, and the packaging efficiency of the packaging composition is higher than that of the prior material, and the packaging is more complete. As shown in fig. 3, the phiX174 phage was packaged separately with regular icosahedron structures containing 6, 18, 30 anchors, and their packaging efficiencies were compared. The left to right bands represent Marker, backbone strand p7560, individual phiX174 long chain, individual icosahedron, icosahedron packaging complex with 30 anchors, icosahedron packaging complex with 18 anchors plus the remaining 12 complementary strands, icosahedron packaging complex with 6 anchors plus the remaining 24 complementary strands. The figure shows that the holding power of 6 anchor points is insufficient and very unstable, meanwhile, some phiX174 remains, the package of 18 anchor points is good, the package efficiency can be saved to a certain extent by adding the remaining complementary chains, the package integrity can be higher by the nano material with 30 anchor points, and the package efficiency is obviously improved.
Specific applications of the double-layer DNA polyhedral frame structure and the packaging assembly thereof related to the present invention and feasibility verification experiments will be specifically discussed later.
Application and feasibility verification of molecular biology operations in confined space
In the experiment, nuclease P1 (Nuclease P1) and mung Bean Nuclease (Mung Bean Nuclease, which are abbreviated as Bean or MBN in experimental illustration) are taken as examples respectively, and the application performance and feasibility of the nano material and the packaging assembly thereof in the nano space molecule operation level are verified. As shown on the left of FIG. 4, these two single-stranded specific nucleases were used to incubate with the phage-like packaging structure, while the negative control was applied with phiX174 alone, which was run for 5min,10min,15min,30min, respectively, to see if they could enter the interior through the regular triangle on the icosahedron surface to exert cleavage.
Wherein, band 1 is the electropherogram result map of Marker, band 2 is the electropherogram result map of phiX174 only, band 3 is the icosahedral electropherogram result map of 30 anchors, band 4 is the electropherogram result map of the artificial phage packaging complex of 30 anchors, band 5 is the electropherogram result map after 5min treatment by adding nucleic acid mung bean nuclease to phiX174, and bands 6, 7, 8, 9 are the electropherogram result maps after 5min,10min,15min,30min treatment by adding mung bean nuclease to the artificial phage packaging complex of 30 anchors, respectively. From the figure, phiX174 in bands 6, 7, 8 and 9 is continuously reduced with the passage of time, and finally phiX174 is completely decomposed, and band 9 is restored to the state of band 3, so that mung bean nuclease can enter the interior to play a role of cleavage through the regular triangle on the surface of the icosahedron. Similarly, as shown in FIG. 4, the effect of using nuclease P1 is the same.
As shown in FIG. 5, the replication increment mode of phiX174 phage is simulated, the anchor point sequence inside the phage-simulated packaging structure is used as a primer, and whether enzyme, raw material and the like subjected to rolling circle replication can enter into the interior through the regular triangle on the surface of the icosahedron to replicate a product phiX174 long chain is observed. Meanwhile, the replication efficiency of RCA under the condition of the presence or absence of an icosahedron structure is compared, namely, the independent phiX174 and the structure of the icosahedron after the phiX174 is packaged are incubated for 15min,30min,1h and 2h at the temperature of 30 ℃, and the running glue is verified. The left to right bands represent markers, individual phiX174 long chain plus 30 primers incubated at 30℃for 0min,15min,30min,1h,2h, respectively, 30 anchor-containing icosahedral packaging complexes incubated at 30℃for 0min,15min,30min,1h,2h, respectively.
As can be seen in FIG. 5, the enzyme, starting material, of the rolling circle replication is indeed able to enter the icosahedron, and finally the product of the rolling circle replication is obtained, and the spinning-like structure of a cluster is visible on the electron microscope image.
The three experiments fully illustrate that the application of the imitated virus DNA polyhedral framework structure and the assembly thereof in the molecular biology operation in the limited space is completely feasible, and the experiment in FIG. 6 further proves that the imitated virus DNA polyhedral framework structure has good application capability in a cell-free in-vitro amplification system and can synthesize the capsid protein of the virus in the cell-free system.
Verification of virus infection imitating capability of composite structure
As shown in FIG. 7, the E.coli C of the phiX174 active type is taken as an example, and after the phiX174 is packed by the regular icosahedron with 6, 18 and 30 anchors extending from the inside, the three types of phage-like packaging structure samples are respectively incubated and cultured with the E.coli C, and meanwhile, compared with the independent phiX174, the bacterial infection experiment by a one-step method is carried out, so that the number of generated plaques is seen. The graph shows that the more the number of anchor points is, the more the number of plaques on the culture dish is, which shows that the twenty-face physical ability assists phiX174 to infect Escherichia coli, and the reproduction and value-added splitting Escherichia coli to generate plaques, thus proving that the imitated virus DNA polyhedral frame structure has excellent application capability in the field of imitated virus infection links.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein, and any modifications, equivalent alterations, improvements, etc., within the spirit and principles of the present invention are to be included within the scope of the present invention.
Claims (9)
1. A virus-like DNA polyhedral frame structure, characterized in that the DNA polyhedral frame structure is a regular icosahedron frame structure, each side is composed of 4DNA double helices, and has:
an outer layer structure, wherein each edge at the vertex is bridged by 5T single-stranded DNA;
the inner layer structure is bridged by 1T between each two sides at the vertex;
the regular icosahedron framework structure comprises 30 sides, each side extending inward or outward from an anchor sequence for downstream grasping of the genome or functionalization of the structure.
2. A method for constructing the virus-like DNA polyhedral framework according to claim 1 by using DNA folding technique, comprising:
step 1: constructing a regular icosahedron framework structure, and presenting a structural schematic diagram by utilizing a 2D/3D image;
step 2: further constructing the edge and the inner and outer layer structures of the regular icosahedron on the basis of the step 1;
step 3: further constructing inner and outer modifiable sites of the regular icosahedron DNA framework structure on the basis of the step 2;
step 4: and (3) carrying out structural assembly and purification on the regular icosahedron DNA framework structure constructed in the step (3).
3. The method of claim 2, wherein step 1 subsequently comprises obtaining the corresponding sequences of the hundreds of staple chains by input of the template chain sequences, and performing sequence synthesis; the step 4 comprises removing unbound single strands of the residual materials and impurities such as dimers and multimers by using an ultracentrifugation and ultrafiltration concentration method.
4. A packaging assembly composition relating to the polyhedral framework of a pseudo-viral DNA according to claim 1, comprising:
an n-icosahedral DNA framework structure having an anchor strand; and
single-or double-stranded circular DNA capable of base complementary pairing with the anchor strand of the regular icosahedron DNA framework structure.
5. The packaging assembly composition according to claim 4, wherein the single-stranded circular DNA is selected from phiX174 genome comprising 5386 bases, and 30 stretches of 20 bases or more are selected as the anchor fragment.
6. A method of constructing the packaged fitment composition of claim 4, comprising the steps of:
step 1: encoding the 30 sides of the imitated viral DNA polyhedral frame structure according to claim 2 in an end-to-end sequence, wherein each side extends out of an anchor chain with the length of 20 bases or more;
step 2: selecting 30 sections of 20 or more base sequences on a single-stranded circular DNA containing 5386 bases as anchor fragments, and performing base complementary pairing with an anchor strand extending out of an icosahedron;
step 3: the packaging composition is purified to remove unbound single strands, as well as some dimers, multimers, etc. from the system.
7. Use of the viroid DNA polyhedral framework according to claim 1 in the manipulation of nano-space molecules.
8. Use of the viroid DNA polyhedral framework according to claim 1 in a cell-free in vitro amplification system.
9. Use of the polyhedral framework of a viroid DNA according to claim 1 in a viroid infection procedure.
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| CN111440610A (en) * | 2020-03-13 | 2020-07-24 | 中国科学院上海高等研究院 | Multicolor fluorescent probe based on DNA nano structure and preparation method and application thereof |
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| CN113278607A (en) * | 2021-04-20 | 2021-08-20 | 南京大学 | Preparation method of circular single-stranded DNA integrated by nucleic acid aptamer and application of circular single-stranded DNA integrated by nucleic acid aptamer in DNA paper folding |
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