CN113004362B - Staple nucleic acid, DNA nano robot and preparation method and application thereof - Google Patents
Staple nucleic acid, DNA nano robot and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a staple nucleic acid, a DNA nano robot and a preparation method and application thereof, wherein the staple nucleic acid comprises a nucleotide sequence shown in any one of SEQ ID No. 1-138; the preparation raw materials of the DNA nano robot comprise a combination of the staple nucleic acids, a bracket chain nucleic acid solution and a buffer solution; in terms of preparation precision, the DNA nano robot can reach about 50nm, has good forming consistency and has huge processing advantages; the DNA nano robot also has the advantages of simple preparation method and good biocompatibility, can be produced in a large scale, and has wider application prospect in the fields of medical materials and medical appliances.
Description
Technical Field
The invention belongs to the technical field of nano materials, and particularly relates to a staple nucleic acid, a DNA nano robot, a preparation method and application thereof.
Background
DNA as a natural material, nanostructures made from DNA may be functionalized with DNA hybridization (e.g., metal nanoparticles or biological components). In addition, DNA nanostructures exhibit better biocompatibility and biodegradability than inorganic nanostructures, and have been demonstrated to have effective cellular internalization and the ability to recognize specific molecules and trigger receptor-mediated cellular pathways on cell membranes after targeting molecule functionalization by DNA nanostructures with greatly reduced cytotoxicity in vitro and in vivo. Based on different size designs and customized functionalization strategies, DNA nanostructures play an indispensable role in the fields of catalysis, biosensing, bioimaging, drug delivery and therapy, etc.
The nanometer robot has great application potential in the field of medical instruments, and as the nanometer robot can enter a narrow space and can actively target specific target tumor cells, the tumor microenvironment contains various biological substance barriers, such as extracellular matrixes and pressure-driven countercurrent; these factors can significantly affect the biodistribution of the drug-loaded nanoparticle and its penetration through the tumor. The problem faced by nanodrug delivery where tumor cells are far from the blood vessels is that nanoparticles relying on free diffusion often cannot reach the depth where tumor cells are located through the biological kinetics and cell targeting moieties. While related work of nano robots is always exploring the possibility of drug delivery, the goal is to develop a nano robot with nano scale, which can be manufactured in a large scale at low cost and can precisely target drug delivery in living body.
At present, the method for preparing the micro-nano robot comprises ultraviolet lithography, laser direct writing, chemical synthesis and biological synthesis, and in the existing research, the technology for preparing the micro-nano robot mainly adopts the ultraviolet lithography technology, the laser direct writing technology, the chemical synthesis method and the biological synthesis method. CN111705299a discloses a preparation method of a nano robot, firstly, wu De alloy film is coated on the surface of a substrate, and Wu De alloy film is etched into protrusions. Then, a raised magnetic film is coated on the surface of the protrusion, and a cavity corresponding to the protrusion is formed in the magnetic film and can be used for carrying medicine; then heating the projections made of Wu De alloy material to separate the magnetic film from the substrate, and finally cutting the magnetic film to obtain the nano robot. The speed of plating the magnetic film on the surface of the substrate through the plating equipment is very high, so that the manufacturing cost of the nano robot can be effectively reduced; the thickness of the magnetic film prepared by the film plating equipment is generally uniform, all components forming the magnetic film can be fully mixed, and the compactness of the magnetic film can be obviously improved. However, the micro-nano robot prepared by the method has limitation on the size, the size of the micro-nano robot can be more than 2 micrometers, and only a two-dimensional robot can be prepared, but a three-dimensional robot with a complex structure can not be prepared.
CN102431966a discloses a tubular porous micrometer motor, its preparation method and application, the preparation steps of the porous micrometer motor of the invention are: anodic oxidation is carried out to prepare an alumina film with a nano-pore array on the surface; depositing a multilayer film with a prestress gradient on the anodic aluminum oxide film; carrying out graphical treatment on the multilayer film; selectively corroding porous anodic aluminum oxide under the multilayer film, and automatically curling the multilayer film into a micrometer pipe with nanometer holes on the pipe wall; transferring the porous micro-tube into the solution to become a micro-motor; the porous micro motor with the special structure has large surface area, higher catalytic efficiency and faster movement speed; the magnetic field can be used for controlling the movement direction of the micrometer motor for transporting the micro-nano level object. The high-speed motion micrometer motor has great application prospect in aspects of drug transportation, biological detection and separation, single cell analysis and the like. However, the technology adopts the principle of photon polymerization, is easily influenced by light diffraction in preparation precision, has relatively poor precision, is greatly influenced by the working performance of machine equipment due to relatively slow preparation engineering, has higher manufacturing cost and is difficult to industrialize.
CN111663995a discloses a chemical energy driven nano engine and a method for providing power and a nano robot. The nano engine provided by the invention comprises: the upper part of the shell is an oil layer, and the lower part of the shell is a water layer; the upper part of the shell is provided with an opening, and the opening is covered with a semipermeable membrane which allows gas to pass through; the nano engine provided by the invention can release a large amount of hydrogen by utilizing the reaction of the metal sodium and water as the driving force of the nano engine, and the movement rate of a nano robot provided with the nano engine can be well ensured. The micro-nano robot prepared by the chemical synthesis method is simple and easy to operate, has relatively low cost and can be produced in a large scale, the scale size of the micro-nano robot can reach tens of nanometers, but the chemical synthesis environment is relatively complex, intermediate products are more, impurities are more after synthesis, the reaction can not be accurately controlled, and the shape consistency is poor.
Therefore, developing a DNA nanorobot that is precisely controllable, has good shape consistency, and is excellent in biocompatibility is an urgent problem in the art to be solved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a staple nucleic acid, a DNA nano robot, a preparation method and application thereof, wherein the DNA nano robot consists of a combination of the staple nucleic acid and a bracket chain nucleic acid, can reach about 50nm in preparation precision, has good forming consistency and has huge processing advantages; the DNA nano robot has strong biocompatibility, simple preparation method, mass production and good application prospect.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides a staple nucleic acid comprising a nucleic acid sequence having a nucleotide sequence as set forth in any one of SEQ ID Nos. 1 to 138.
Wherein, the nucleotide sequences of SEQ ID No.1 to 138 are as follows:
SEQ ID No.1：GGCAAAGAGGCGCCCGGATTGCGATTGAATACCAGAGGG；
SEQ ID No.2：CAAGCGGTCCACGCTGGTTTGC；
SEQ ID No.3：ACTGCGTTCAGAACATGTTTATCAT；
SEQ ID No.4：CACCACCTTAGCGTCATTTGGGAAAGACAAAAATCCCGCC；
SEQ ID No.5：CGAGGCTGGGCTCGAGCTGATACAGGAGCCAGGGGCATCGTA；
SEQ ID No.6：CCAGCATTGGAAAGTTGAGTAAGGCTGATAGGTGTCACCCTC；
SEQ ID No.7：CCAACGCGCTCACATAATAAA；
SEQ ID No.8：GGCACCGCTAAAAATAACCGGTTG；
SEQ ID No.9：AGACTGATAAATCTGAATATCCTGGTTGCGGCCCTG；
SEQ ID No.10：CACCCTCAAGCCCTTAGTA；
SEQ ID No.11：TATTTAACTCATATCGCAAGGTTTCATTGCAACTAAATGCTT；
SEQ ID No.12：ATATTCATTGCAAGCGGAGTACGAACTAACTCATAA；
SEQ ID No.13：ATTTGTTTAGCTTTAACGTAA；
SEQ ID No.14：GCCAAAATAACGACGGCTAAGCAATATG；
SEQ ID No.15：ATGCCACCAACTTTGGCTGCGTTGGGATTACAGTCCAAT；
SEQ ID No.16：GCGGTTTGCGTATTGGTTTGGAACTGCAGGCGGATAAGGTACCGT；
SEQ ID No.17：CAACCTATAAGGAGTAATTCATTACATCAGATGTTT；
SEQ ID No.18：CATTCAACCGTCACCGACCGTATAAATT；
SEQ ID No.19：TCGTCAAACCCATTGCCGTCTGAGTGTTCGTGGACT；
SEQ ID No.20：TAAACAGGAATCGGGAAC；
SEQ ID No.21：AACATTAAGAAAACAGTTGAGCCTTTTATTA；
SEQ ID No.22：CTAACTCACATTAATTGGTGCTTGGAGGTCATTTCGCAA；CCTTAATTCA；
SEQ ID No.23：ATAGGCTGAAAGATGCAATATTGTATGTTAAATC；
SEQ ID No.24：GAGCCGCCGTAGCGACATCACCAGAAGGGCGATAGCC；CGACAGTGAGA；
SEQ ID No.25：AAGAAGGAAGCAATTGATAATTACCTCGTCTAATCT；
SEQ ID No.26：CCCAGCAGGCGAACACCCTCAG；
SEQ ID No.27：CCGGGCATCCGAGTACCCTGACGAGAAA；
SEQ ID No.28：AAATTATTCATTACTGCCTATT；
SEQ ID No.29：TCAATCATGGCCGGTAAATCGGGATACATTTTTGCGACTATT；
SEQ ID No.30：TTACCCACTTAGCCAAATCAGTAAAACCAGCTTTC；
SEQ ID No.31：ACAACGGGGATAGAGTATAGCAAAAGAAAAAACCGT；
SEQ ID No.32：GTCATTGGCCGGAGATAAATCAAAAGGTGAAGCAATTCAAAT；
SEQ ID No.33：CAGCGAAAGACAGCATCGGAACTTTTGCATCAGCAAAAAAAGA；
SEQ ID No.34：TTGTCAACTCAGGAGACAAGGCAGCTG；
SEQ ID No.35：CTATGATACGATTAAGTAAAAACAAAAAGGGTCAATCAAAACGAAAGA；
SEQ ID No.36：CGGCTAGCAGGGAGTGAATTCACGTTTTTTCTGCATTCC；
SEQ ID No.37：GTTTTGTCGTCTTTCCGGCTCCATTATTCATGC；
SEQ ID No.38：ATCGCGTCGTTTAGAGCAACACTAT；
SEQ ID No.39：ACAGACGAGCCACATCACCGAATCG；
SEQ ID No.40：CAACTATGAATTAATGGTCTCAGAGCGTCACGACCCTCAGGA；
SEQ ID No.41：GTGCGGGCATAGGGGCCTTGAAGAGTCCACTACCGGAAACTTTGATG；
SEQ ID No.42：AACACTATGTTAAATCAATCATTGATGCAGTTGCAA；
SEQ ID No.43：AGTAGTAGCATTTGGTTGTGAA；
SEQ ID No.44：ATTAATGAATCGGTTCCGTCGGTGCGCGCCATTCA；
SEQ ID No.45：GGCACGAATCTCTTCGCATTTCAAATTT；
SEQ ID No.46：AAGTACAGACCACGTTAATAAAGATGGAAGATAAAAATGTCGATCG；
SEQ ID No.47：CAAAGCGCGACCTTCAACAGAGAATCAGCGAGTA；
SEQ ID No.48：TAACGCTTTCAACTATATAGCAAAATCAGTG；
SEQ ID No.49：ATTTACGCTTTTCCCAATAAAGCAGACAGTCGGAACAGAATACACTAA；
SEQ ID No.50：GTTATCCGCTCACAATTCCTGTTTCCTCCAGA；
SEQ ID No.51：GGACTAATTCGGTTGATATTGCGAGCTAAACAAACT；
SEQ ID No.52：GATTTTATAATACAAGAGAACGGGGTCCAGC；
SEQ ID No.53：TGAATAAATAAATAATTAATCCTGAGACTCCGTGG；
SEQ ID No.54：TATTCACAAGTCACCCTCAG；
SEQ ID No.55：AATGAAGAAAATTATTAAGACAGTGCCTTGA；
SEQ ID No.56：TCGGAACCTAAAAGGAGCCT；
SEQ ID No.57：CTTCAAAGCGAAGTGTGAAATT；
SEQ ID No.58：GCAAACACGTTCTAAAGAATTTTTCATTGGATTAGTTAAGAG；
SEQ ID No.59：CATAACCCTTTTAATTCGAG；
SEQ ID No.60：CAATAGGCCAGTACAACT；
SEQ ID No.61：CTTCACCGCCTGGGCAAAGGGCGAGCCAGACTGTA；
SEQ ID No.62：ATGACCGATAGCGGTAGA；
SEQ ID No.63：CCCAAGGTGAATTTTCGGTCATAGCCCCCTTAT；
SEQ ID No.64：CCGTAATCAACCCTCAGCGCCACC；
SEQ ID No.65：CCCTCAGACCATCGAAGCAAGGTTAAAGAAGTTCCAGGCGCCAGG；
SEQ ID No.66：CGTCATAGTTAGCGTCCTCAGAGTACCGCAATCCTGT；
SEQ ID No.67：AAACAGCTCGCTGTCTGAATCACCAGACTCCCTCA；
SEQ ID No.68：GCGCGTTTTTCACCGGAAGGTTGAG；
SEQ ID No.69：ACCACCCATACATGGCTTTTTACCATCAAGTTTCCATTA；
SEQ ID No.70：TACGAGCCGGAAGCATCGAATTCGCAGGTCATGGGGCGTGAGATTTCAG；
SEQ ID No.71：ATAGTCATTTTGCCACATT；
SEQ ID No.72：CTATCACCAATTAGAGCAGTGCCCGCA；
SEQ ID No.73：ACCGTGCAGTCGGATTGTCTGGA；
SEQ ID No.74：AAAAGAATCAAGTTTGCCTGGAACCGCGCCGCCG；
SEQ ID No.75：ACAACCCTCTGCCAGAGCTTTCCTTATGACCCGAGCTAAA；
SEQ ID No.76：ATAATCAAAGATTCTTATGACGTAGATTTAATTGCAAAAATC；
SEQ ID No.77：AAAATCCCCCTCAAAGTACGGAATCGGCTGACGCATTGGGGAGAG；
SEQ ID No.78：AACCTGTCGTGCCAGCTCTGACCTAATGCTGTATTCTGTCAGGAACCTT；
SEQ ID No.79：TTAATTGTATCGGTTTGGGATCACAAATAAGACGATCTTTTCAT；
SEQ ID No.80：GCAGGTCAATCCTCACAGTTATGAAACAAGGTTTAACCGC；
SEQ ID No.81：AGAACCACTTACCGAAGTTTTAAGGATTTTGATATACAAGCC；
SEQ ID No.82：CACCAGAAAATTCTACTAAT；
SEQ ID No.83：ATTTAGTTAAGAACCATTATTGTACCTGGGTAACAGCTTTCC；
SEQ ID No.84：GGTGCCTAAGGAGGATCTTTA；
SEQ ID No.85：AAAGGCCGCGAGGGTAGCAA；
SEQ ID No.86：TATCCATATAAATCTAGGCGC；
SEQ ID No.87：TTCATGCGCACGACTTAAGCTTCTAAGAACAT；
SEQ ID No.88：TAGCGTTTGCCATTGGCCTTGA；
SEQ ID No.89：GGTAATCCCATCAATAAAGCCAATAACCGCTCCTTAGCGGAT；
SEQ ID No.90：CAGGAAGGCCTGAGCGGGAGAATTCCCAAGCTCAAAACGAGA；
SEQ ID No.91：AGATCTACAAAGGACCGTAATG；
SEQ ID No.92：ACGCCATCTCTGGTGCTGCAAGGCCGACAGTGGTGTAATGAGTCGGGA；
SEQ ID No.93：GACGACGACTAAATGTGGATGAAC；
SEQ ID No.94：ATCAACATAGTATCGGGTTGTAAAACCCCGCTATAAAGACTGAGTGAG；
SEQ ID No.95：TAGTTGCCATAACAAGCGTCTCAGAGCAACCGCCA；
SEQ ID No.96：CGGGCAACAGCTGATTTTATAAATCCCGGAAGACTCCTATTTTTTTCTT；
SEQ ID No.97：CCAAGGGTATATCATACAAAGTACAACG；
SEQ ID No.98：CATCAAGGAACCGGTAGGTAGAAAAGCCAATAGGA；
SEQ ID No.99：TAAAGGACATAAAATACGTA；
SEQ ID No.100：ATTTTCACCTGTAGTATGG；
SEQ ID No.101：ATACAGGTTTGCTCAGTACTCGAATAGAATGAC；
SEQ ID No.102：TTCGAGGTTAAAGCCAGAATGACAGGACCAGAGC；
SEQ ID No.103：ACAAGAAAGACGGTGAGAAAATGTACCTTCGCGTC；
SEQ ID No.104：GGCTGCGCAAAATTTTTAAGCAAA；
SEQ ID No.105：ATGAGGGCCCACGGCCGACAAAGGAAAGTTTCCTGAGTT；
SEQ ID No.106：GAAGCCCTAAAAAGAATTA；
SEQ ID No.107：AGATCGCACCTGTAGCTAGCATG；
SEQ ID No.108：TGTAACTGACTACGAAGGCAC；
SEQ ID No.109：GAACAAACAGATGGGCTGGATGTTTGTCCTTAGTCATAGCACACAACA；；
SEQ ID No.110：TTCAACCAACTAGCGGGGTAGTGTACTTACCATTTAGCAGCA；
SEQ ID No.111：GGTAATTACCCTGGATGGCTGGGCTTAAGAGTGACT；
SEQ ID No.112：TGCATCAAGGCTTATACA；
SEQ ID No.113：GGATAGGTCACGTTGGTGTGGCGGATTGCTATCAG；
SEQ ID No.114：GTGGTTTTTCTTTTCACGATAGGGTGAGAGGGAGGATTAAAGGAACCGA；
SEQ ID No.115：AGCGAGAAAAGAAGAGTACCCCCGG；
SEQ ID No.116：CGACGAGAAAGACACTCCAATAATCATGGTGCTGAA；
SEQ ID No.117：AGGTCTTAGTAAATTGAG；
SEQ ID No.118：AACCGCCACAACGATCTAAA；
SEQ ID No.119：TGCCGGAAACCAGGCAAAGTTTTTAACCCCAAAAA；
SEQ ID No.120：GTCAGGCTTCAGAGGCTTTGA；
SEQ ID No.121：AAGATTTACCAGCGAACGACCTGTAAGGATG；
SEQ ID No.122：CCCAGCGAAATCCTGCTCAGGTTTAACAAAAGCCAAAAT；
SEQ ID No.123：ATATGCTCCCATCTTTGACC；
SEQ ID No.124：AACGGGGATGAACGGTCAAGAACCATTGTAAACGTTACC；
SEQ ID No.125：CGCGAACGCCTGGGCTTGGTAAATATAGTAACCAGA；
SEQ ID No.126：TTCGCATTACTGTTGGGAAGGGCACCAATGAAAGATATTTTGTTAAAA；
SEQ ID No.127：GAGGGAAGGTAAATATTGACGG；
SEQ ID No.128：CCCTGAGAGGGTTCCGATACTC；
SEQ ID No.129：ATATGTGTCGATTATACCAAG；
SEQ ID No.130：CTCACTGCCCGCTTTCCAGTAAACATAGAGCTTAGTTTGACTGGCCTTG；
SEQ ID No.131：CCAACGTCATAGCACCATGGTAATTTCCAGTCGATATAAGACTTTTTC；
SEQ ID No.132：AGCTCATCGCCATTCCAGCTGGAAGCAACTTCCGAACTGC；
SEQ ID No.133：GAGATTTGGCTATTTTTGAG；
SEQ ID No.134：GTGTAAAGCCTGGGTAAATGTCCCCCATTTGAGGG；
SEQ ID No.135：TGGCCTTCTCCAGCCGCCAGGGTCGCCCTGGCTACGTGCGTTGCG；
SEQ ID No.136：AGGTGAAAGCTCCAATTGCT；
SEQ ID No.137：CCATCTGTCGAAAGGGTACTTTTGTAA；
SEQ ID No.138：AATCAAAACATCGGCATTATCACCGATTGAGGTTGATGGTAGTTGCAG。
in a second aspect, the present invention provides a DNA nanorobot whose preparation raw materials include a combination of staple nucleic acids, a rack chain nucleic acid solution, and a buffer as described in the first aspect.
The scaffold strand nucleic acid solution may be selected from a scaffold solution.
Preferably, the DNA nanorobot is C-shaped.
As a preferable technical scheme of the invention, the DNA nanorobot provided by the invention is C-shaped, and the structural schematic diagram is shown in figure 1.
Preferably, the molar ratio of the staple nucleic acid to the scaffold strand nucleic acid in each of the solution of scaffold strand nucleic acids is 1 (0.05-1.5), e.g. 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:1, 1:1.1, 1:1.2, 1:1.3 or 1:1.4, etc.
As a preferable technical scheme of the invention, the molar ratio of each staple nucleic acid to the bracket chain nucleic acid in the bracket chain nucleic acid solution in the DNA nano robot is 1 (0.05-1.5), so that the prepared DNA nano robot is accurately controllable, and on one hand, if the dosage of the staple nucleic acid is too high, the waste of nucleic acid staple materials can be caused; on the other hand, if the amount of staple nucleic acid is too low, the shape integrity of the synthetically set DNA nanorobot may be poor.
Preferably, the buffer comprises a combination of ethylenediamine tetraacetic acid, magnesium chloride, sodium chloride and Tris.
Preferably, the molar ratio of ethylenediamine tetraacetic acid to magnesium chloride is 1 (12-13), such as 1:12.1, 1:12.2, 1:12.3, 1:12.4, 1:12.5, 1:12.6, 1:12.7, 1:12.8, or 1:12.9, etc.
Preferably, the molar ratio of ethylenediamine tetraacetic acid to sodium chloride is 1 (12-13), such as 1:12.1, 1:12.2, 1:12.3, 1:12.4, 1:12.5, 1:12.6, 1:12.7, 1:12.8 or 1:12.9, etc.
Preferably, the molar ratio of ethylenediamine tetraacetic acid to sodium chloride is 1 (4-6), such as 1:4.2, 1:4.4, 1:4.6, 1:4.8, 1:5, 1:5.2, 1:5.4, 1:5.6, or 1:5.8, etc.
Preferably, the Tris has a pH of 7 to 8, such as 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9, and specific values between the above values, are limited in space and for the sake of brevity the invention is not intended to be exhaustive of the specific values comprised in the range.
In a third aspect, the present invention provides a method for preparing a DNA nanorobot according to the second aspect, the method comprising the steps of:
(1) Dissolving staple nucleic acids comprising nucleotide sequences shown in SEQ ID No. 1-138 in water to obtain staple nucleic acid solutions comprising nucleotide sequences shown in SEQ ID No. 1-138;
(2) And (3) reacting the staple nucleic acid solution, the bracket chain nucleic acid solution and the buffer solution which are obtained in the step (1) and contain the nucleotide sequences shown in SEQ ID No. 1-138 to obtain the DNA nano robot.
Preferably, the molar percentage of staple nucleic acid in each of said staple nucleic acid solutions of step (1) is 100-300 nM, e.g. 120nM, 140nM, 160nM, 180nM, 200nM, 220nM, 240nM, 260nM, 280nM or 300nM, and specific point values between the above-mentioned point values, limited in length and for the sake of brevity the invention is not exhaustive list of the specific point values comprised in said range.
Preferably, the reaction of step (2) is carried out under reduced temperature conditions.
Preferably, the cooling method comprises the following steps: the system of 85-95deg.C (for example, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃, etc.) is kept for 5-15 min (for example, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃, etc.), the temperature is reduced to 75-85 ℃ (76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, etc.), the temperature is reduced to 55-65 ℃ (for example, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, etc.), the temperature is reduced to 15-25 ℃ (for example, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, etc.), the temperature is reduced to 15-25 ℃ for the third time.
Preferably, the cooling rate of the first cooling is 5 to 15 ℃/min, for example, 6 ℃/min, 7 ℃/min, 8 ℃/min, 9 ℃/min, 10 ℃/min, 11 ℃/min, 12 ℃/min, 13 ℃/min or 14 ℃/min, and the specific point values between the above point values, which are limited in space and for the sake of brevity, the present invention is not exhaustive to list the specific point values included in the range.
Preferably, the cooling rate of the second cooling is 5 to 15 ℃/min, for example, 6 ℃/min, 7 ℃/min, 8 ℃/min, 9 ℃/min, 10 ℃/min, 11 ℃/min, 12 ℃/min, 13 ℃/min or 14 ℃/min, and the specific point values between the above point values, which are limited in space and for brevity, the present invention is not exhaustive of the specific point values included in the range.
Preferably, the cooling rate of the third cooling is 5 to 15 ℃/h, for example, 6 ℃/min, 7 ℃/min, 8 ℃/min, 9 ℃/min, 10 ℃/min, 11 ℃/min, 12 ℃/min, 13 ℃/min or 14 ℃/min, and the specific point values among the above point values, which are limited in space and for the sake of brevity, the present invention is not exhaustive to list the specific point values included in the range.
Preferably, the cooling is performed under conditions of the metal bath.
Preferably, the preparation method comprises the following steps:
(1) Respectively dissolving staple nucleic acid containing nucleotide sequences shown in SEQ ID No. 1-138 in water to respectively obtain staple nucleic acid solutions containing the nucleotide sequences shown in SEQ ID No. 1-138 with the mole percentage content of 100-300 nM;
(2) Reacting the staple nucleic acid solution, the bracket chain nucleic acid solution and the buffer solution which are obtained in the step (1) and contain the nucleotide sequences shown in SEQ ID No. 1-138 under the cooling condition of a metal bath to obtain the DNA nano robot; the cooling method comprises the following steps: maintaining the temperature of the system at 85-95 ℃ for 5-15 min, cooling to 75-85 ℃ for the first time, adjusting the cooling rate, cooling to 55-65 ℃ for the second time, adjusting the cooling rate, and cooling to 15-25 ℃ for the third time, thereby completing the cooling; the cooling rate of the first cooling is 5-15 ℃/min; the cooling rate of the second cooling is 5-15 ℃/min; the cooling rate of the third cooling is 5-15 ℃/h.
In a fourth aspect, the present invention provides a DNA nanorobot as described in the second aspect for use in medical materials or medical devices.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a staple nucleic acid, and further a DNA nano robot which can reach about 50nm in preparation precision and has good forming consistency, and has great advantages in nano-level processing for the prior art; the DNA nano robot mainly comprises nucleic acid, the nucleic acid is used as a main component substance of organisms, and compared with ultraviolet photoresist, the chemical substance has stronger biocompatibility, so that the prepared DNA nano robot medical material and medical instrument field have wider application prospect.
(2) The DNA nano robot provided by the invention has precise controllability, can carry out precise and multi-site modification on DNA through the nucleotide sequence in the staple nucleic acid in the DNA robot so as to be convenient for connection with materials, antibodies and magnetic beads thereof, and can carry out functional group transformation on the DNA nano robot so as to gradually lead the DNA nano robot to be applied, and the DNA nano robot has fewer single impurities and can be produced in a large scale.
Drawings
FIG. 1 is a schematic diagram of a C-type DNA nanorobot according to the present invention;
FIG. 2 is a transmission electron microscope image at x100k times of the DNA nanorobot provided in example 139;
fig. 3 is a transmission electron microscope image at x12.0 k-times of the DNA nanorobot provided in example 139.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Examples 1 to 138
A staple nucleic acid comprising nucleotide sequences having the nucleotide sequences shown in SEQ ID nos. 1 to 138, respectively;
the preparation method comprises the following steps: model C was simulated using the canAno software, and the nucleotide sequences shown in SEQ ID Nos. 1 to 138 were derived, and were prepared according to the nucleotide sequences in the company Shanghai, inc., to obtain staple nucleic acids having the nucleotide sequences shown in SEQ ID Nos. 1 to 138, respectively.
Example 139
A DNA nanorobot, the preparation raw materials of which include a combination comprising staple nucleic acid having the nucleotide sequence shown in SEQ ID No. 1-138 (examples 1-138), a solution of a bracket chain nucleic acid (scaffold solution, tilibit, p7560, 100nM of bracket chain nucleic acid) and a buffer (1 mM EDTA, 12.5mM MgCl, 2.5mM NaCl, 5mM Tris (pH=7.9));
the preparation method comprises the following steps:
(1) The staple nucleic acids comprising the nucleotide sequences shown in SEQ ID Nos. 1 to 138 obtained in examples 1 to 138 were dissolved in water, respectively, to obtain staple nucleic acid solutions each having a molar percentage of 200 nM;
(2) Reacting 55.2 mu L of all the staple nucleic acid solution obtained in the step (1), 40 mu L of the rack chain nucleic acid solution and the buffer solution in a metal bath (ensuring the staple nucleic acid) to obtain the DNA nano robot; the conditions of the metal bath were set as follows: and (3) preserving heat at the system at 90 ℃ for 10min, cooling to 80 ℃ according to 10 ℃/min, cooling to 60 ℃ according to 10 ℃/min, cooling to 20 ℃ according to 10 ℃/h, and completing the cooling.
Example 140
A DNA nanorobot, the preparation raw materials of which include a combination comprising staple nucleic acid having the nucleotide sequence shown in SEQ ID No. 1-138 (examples 1-138), a solution of a bracket chain nucleic acid (scaffold solution, tilibit, p7560, bracket chain nucleic acid 100 nM) and a buffer (1 mM EDTA, 12mM MgCl, 12mM NaCl, 4mM Tris (pH=7.9));
the preparation method comprises the following steps:
(1) The staple nucleic acids comprising the nucleotide sequences shown in SEQ ID Nos. 1 to 138 obtained in examples 1 to 138 were dissolved in water, respectively, to obtain staple nucleic acids each having a molar percentage of 100 nM;
(2) Reacting 55.2 mu L of all the staple nucleic acid solution obtained in the step (1), 40 mu L of the bracket chain nucleic acid solution and the buffer solution in a metal bath to obtain the DNA nano robot; the conditions of the metal bath were set as follows: and (3) preserving heat for 15min at the system of 85 ℃, cooling to 75 ℃ according to 5 ℃/min, cooling to 55 ℃ according to 15 ℃/min, cooling to 15 ℃ according to 15 ℃/h, and completing the cooling.
Example 141
A DNA nanorobot, the preparation raw materials of which include a combination comprising staple nucleic acid having the nucleotide sequence shown in SEQ ID No. 1-138 (examples 1-138), a solution of a bracket chain nucleic acid (scaffold solution, tilibit, p7560, 120nM of bracket chain nucleic acid) and a buffer (1 mM EDTA, 13mM MgCl, 13mM NaCl, 6mM Tris (pH=7.9));
the preparation method comprises the following steps:
(1) The staple nucleic acids comprising the nucleotide sequences shown in SEQ ID Nos. 1 to 138 obtained in examples 1 to 138 were dissolved in water, respectively, to obtain staple nucleic acids each having a molar percentage of 300 nM;
(2) Reacting 55.2 mu L of all the staple nucleic acid solution obtained in the step (1), 40 mu L of the bracket chain nucleic acid solution and the buffer solution in a metal bath to obtain the DNA nano robot; the conditions of the metal bath were set as follows: and (3) preserving the temperature of the system at 95 ℃ for 5min, cooling to 85 ℃ according to 15 ℃/min, cooling to 65 ℃ according to 5 ℃/min, cooling to 25 ℃ according to 5 ℃/h, and completing the cooling.
Performance test:
morphology observation:
(1) ddH is to 2 O is heated to boiling, and ddH is removed after boiling is continued for 2min 2 Oxygen of O; (2) weigh 0.1g UFO into a 10mL centrifuge tube and add 5mL hot ddH 2 O to UFo powder, tightly covering the cover, and tightly wrapping with tinfoil paper to enable the powder to be in a light-shielding state and vortex for 10min; (3) filtering the vortex dyeing liquid in a 0.2 mu m filter to remove impurities; (4) subpackaging into 1mL centrifuge tubes, and centrifuging at 13000rpm for 5min;5.5 mu L of 5M NaOH solution into the dye liquor, and swirling for 2-3 min to mix uniformly; (6) centrifuging at 13000rpm for 5min, collecting supernatant, and performing ultrasonic treatment for 5min to obtain fresh negative dye liquor; (7) placing a 300-mesh carbon film copper net into a plasma for treatment so as to enhance the hydrophilicity of the carbon film copper net; (8) a piece of copper net treated by plasma is clamped by self-locking tweezers and placed on a tidied tabletop, a 3uL sample is vertically dripped on the copper net by a pipetting gun, and the copper net is kept stand for 3-4 min; (9) sucking the redundant sample with filter paper, standing for 1min, airing to a dry and undried state, vertically suspending 25 mu L of negative dye liquor by using a pipetting gun, and dyeing for 30s (sucking the dye liquor with the filter paper at the time of 25 s); and was examined using HT7700 transmission electron microscopy.
The transmission electron microscope images of the DNA nanorobot prepared in example 139 at (x 100 k) times and (x 12.0 k) times are shown in FIG. 2 and FIG. 3, respectively, and it can be seen from FIG. 2 that the DNA nanorobot prepared in example 139 is C-shaped and has a radius of about 50nm, and it can be seen from FIG. 3 that the DNA nanorobot prepared in example 139 has uniform size and is C-shaped. The method has the advantages that the preparation precision can reach about 50nm, the forming consistency is good, and the method has great advantages on nano-scale processing for the prior art; the DNA nano robot mainly comprises nucleic acid, the nucleic acid is used as a main component substance of organisms, and compared with ultraviolet photoresist, the chemical substance has stronger biocompatibility, so that the prepared DNA nano robot medical material and medical instrument field have wider application prospect.
The applicant states that the present invention is illustrated by the above examples for staple nucleic acids, DNA nanorobots, and methods of making and using the same, but the present invention is not limited to, i.e., does not mean that the present invention must rely on the above process steps to be practiced. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
SEQUENCE LISTING
<110> university of south science and technology
<120> a staple nucleic acid, DNA nanorobot, and preparation method and application thereof
<130> 2021-2-23
<160> 138
<170> PatentIn version 3.3
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ggcaaagagg cgcccggatt gcgattgaat accagaggg 39
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caagcggtcc acgctggttt gc 22
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actgcgttca gaacatgttt atcat 25
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caccacctta gcgtcatttg ggaaagacaa aaatcccgcc 40
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cgaggctggg ctcgagctga tacaggagcc aggggcatcg ta 42
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ccagcattgg aaagttgagt aaggctgata ggtgtcaccc tc 42
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ccaacgcgct cacataataa a 21
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ggcaccgcta aaaataaccg gttg 24
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agactgataa atctgaatat cctggttgcg gccctg 36
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caccctcaag cccttagta 19
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tatttaactc atatcgcaag gtttcattgc aactaaatgc tt 42
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atattcattg caagcggagt acgaactaac tcataa 36
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atttgtttag ctttaacgta a 21
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gccaaaataa cgacggctaa gcaatatg 28
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atgccaccaa ctttggctgc gttgggatta cagtccaat 39
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gcggtttgcg tattggtttg gaactgcagg cggataaggt accgt 45
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caacctataa ggagtaattc attacatcag atgttt 36
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<400> 18
cattcaaccg tcaccgaccg tataaatt 28
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<211> 36
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<400> 19
tcgtcaaacc cattgccgtc tgagtgttcg tggact 36
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<400> 20
taaacaggaa tcgggaac 18
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<400> 21
aacattaaga aaacagttga gccttttatt a 31
<210> 22
<211> 49
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<400> 22
ctaactcaca ttaattggtg cttggaggtc atttcgcaac cttaattca 49
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<400> 23
ataggctgaa agatgcaata ttgtatgtta aatc 34
<210> 24
<211> 48
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<400> 24
gagccgccgt agcgacatca ccagaagggc gatagcccga cagtgaga 48
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<211> 36
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<400> 25
aagaaggaag caattgataa ttacctcgtc taatct 36
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cccagcaggc gaacaccctc ag 22
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ccgggcatcc gagtaccctg acgagaaa 28
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<400> 28
aaattattca ttactgccta tt 22
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<400> 29
tcaatcatgg ccggtaaatc gggatacatt tttgcgacta tt 42
<210> 30
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<400> 30
ttacccactt agccaaatca gtaaaaccag ctttc 35
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<400> 31
acaacgggga tagagtatag caaaagaaaa aaccgt 36
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gtcattggcc ggagataaat caaaaggtga agcaattcaa at 42
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cagcgaaaga cagcatcgga acttttgcat cagcaaaaaa aga 43
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ttgtcaactc aggagacaag gcagctg 27
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<400> 35
ctatgatacg attaagtaaa aacaaaaagg gtcaatcaaa acgaaaga 48
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<400> 36
cggctagcag ggagtgaatt cacgtttttt ctgcattcc 39
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gttttgtcgt ctttccggct ccattattca tgc 33
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atcgcgtcgt ttagagcaac actat 25
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acagacgagc cacatcaccg aatcg 25
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caactatgaa ttaatggtct cagagcgtca cgaccctcag ga 42
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gtgcgggcat aggggccttg aagagtccac taccggaaac tttgatg 47
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<400> 42
aacactatgt taaatcaatc attgatgcag ttgcaa 36
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<400> 43
agtagtagca tttggttgtg aa 22
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<212> DNA
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<400> 44
attaatgaat cggttccgtc ggtgcgcgcc attca 35
<210> 45
<211> 28
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<400> 45
ggcacgaatc tcttcgcatt tcaaattt 28
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<400> 46
aagtacagac cacgttaata aagatggaag ataaaaatgt cgatcg 46
<210> 47
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<400> 47
caaagcgcga ccttcaacag agaatcagcg agta 34
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<400> 48
taacgctttc aactatatag caaaatcagt g 31
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atttacgctt ttcccaataa agcagacagt cggaacagaa tacactaa 48
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<400> 50
gttatccgct cacaattcct gtttcctcca ga 32
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ggactaattc ggttgatatt gcgagctaaa caaact 36
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gattttataa tacaagagaa cggggtccag c 31
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tgaataaata aataattaat cctgagactc cgtgg 35
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tattcacaag tcaccctcag 20
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aatgaagaaa attattaaga cagtgccttg a 31
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tcggaaccta aaaggagcct 20
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cttcaaagcg aagtgtgaaa tt 22
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gcaaacacgt tctaaagaat ttttcattgg attagttaag ag 42
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cataaccctt ttaattcgag 20
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caataggcca gtacaact 18
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cttcaccgcc tgggcaaagg gcgagccaga ctgta 35
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atgaccgata gcggtaga 18
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cccaaggtga attttcggtc atagccccct tat 33
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ccgtaatcaa ccctcagcgc cacc 24
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ccctcagacc atcgaagcaa ggttaaagaa gttccaggcg ccagg 45
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cgtcatagtt agcgtcctca gagtaccgca atcctgt 37
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aaacagctcg ctgtctgaat caccagactc cctca 35
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<400> 68
gcgcgttttt caccggaagg ttgag 25
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accacccata catggctttt taccatcaag tttccatta 39
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tacgagccgg aagcatcgaa ttcgcaggtc atggggcgtg agatttcag 49
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atagtcattt tgccacatt 19
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ctatcaccaa ttagagcagt gcccgca 27
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accgtgcagt cggattgtct gga 23
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aaaagaatca agtttgcctg gaaccgcgcc gccg 34
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acaaccctct gccagagctt tccttatgac ccgagctaaa 40
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ataatcaaag attcttatga cgtagattta attgcaaaaa tc 42
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aaaatccccc tcaaagtacg gaatcggctg acgcattggg gagag 45
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aacctgtcgt gccagctctg acctaatgct gtattctgtc aggaacctt 49
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ttaattgtat cggtttggga tcacaaataa gacgatcttt tcat 44
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gcaggtcaat cctcacagtt atgaaacaag gtttaaccgc 40
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agaaccactt accgaagttt taaggatttt gatatacaag cc 42
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caccagaaaa ttctactaat 20
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atttagttaa gaaccattat tgtacctggg taacagcttt cc 42
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<400> 84
ggtgcctaag gaggatcttt a 21
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<400> 85
aaaggccgcg agggtagcaa 20
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tatccatata aatctaggcg c 21
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ttcatgcgca cgacttaagc ttctaagaac at 32
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<400> 88
tagcgtttgc cattggcctt ga 22
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<400> 89
ggtaatccca tcaataaagc caataaccgc tccttagcgg at 42
<210> 90
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<400> 90
caggaaggcc tgagcgggag aattcccaag ctcaaaacga ga 42
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<400> 91
agatctacaa aggaccgtaa tg 22
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<400> 92
acgccatctc tggtgctgca aggccgacag tggtgtaatg agtcggga 48
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<400> 93
gacgacgact aaatgtggat gaac 24
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atcaacatag tatcgggttg taaaaccccg ctataaagac tgagtgag 48
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<400> 95
tagttgccat aacaagcgtc tcagagcaac cgcca 35
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<400> 96
cgggcaacag ctgattttat aaatcccgga agactcctat ttttttctt 49
<210> 97
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<400> 97
ccaagggtat atcatacaaa gtacaacg 28
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<400> 98
catcaaggaa ccggtaggta gaaaagccaa tagga 35
<210> 99
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<400> 99
taaaggacat aaaatacgta 20
<210> 100
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<400> 100
attttcacct gtagtatgg 19
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<400> 101
atacaggttt gctcagtact cgaatagaat gac 33
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ttcgaggtta aagccagaat gacaggacca gagc 34
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<400> 103
acaagaaaga cggtgagaaa atgtaccttc gcgtc 35
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<400> 104
ggctgcgcaa aatttttaag caaa 24
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atgagggccc acggccgaca aaggaaagtt tcctgagtt 39
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<400> 106
gaagccctaa aaagaatta 19
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<400> 107
agatcgcacc tgtagctagc atg 23
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<400> 108
tgtaactgac tacgaaggca c 21
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gaacaaacag atgggctgga tgtttgtcct tagtcatagc acacaaca 48
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<400> 110
ttcaaccaac tagcggggta gtgtacttac catttagcag ca 42
<210> 111
<211> 36
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<400> 111
ggtaattacc ctggatggct gggcttaaga gtgact 36
<210> 112
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<400> 112
tgcatcaagg cttataca 18
<210> 113
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<213> artificial sequence
<400> 113
ggataggtca cgttggtgtg gcggattgct atcag 35
<210> 114
<211> 49
<212> DNA
<213> artificial sequence
<400> 114
gtggtttttc ttttcacgat agggtgagag ggaggattaa aggaaccga 49
<210> 115
<211> 25
<212> DNA
<213> artificial sequence
<400> 115
agcgagaaaa gaagagtacc cccgg 25
<210> 116
<211> 36
<212> DNA
<213> artificial sequence
<400> 116
cgacgagaaa gacactccaa taatcatggt gctgaa 36
<210> 117
<211> 18
<212> DNA
<213> artificial sequence
<400> 117
aggtcttagt aaattgag 18
<210> 118
<211> 20
<212> DNA
<213> artificial sequence
<400> 118
aaccgccaca acgatctaaa 20
<210> 119
<211> 35
<212> DNA
<213> artificial sequence
<400> 119
tgccggaaac caggcaaagt ttttaacccc aaaaa 35
<210> 120
<211> 21
<212> DNA
<213> artificial sequence
<400> 120
gtcaggcttc agaggctttg a 21
<210> 121
<211> 31
<212> DNA
<213> artificial sequence
<400> 121
aagatttacc agcgaacgac ctgtaaggat g 31
<210> 122
<211> 39
<212> DNA
<213> artificial sequence
<400> 122
cccagcgaaa tcctgctcag gtttaacaaa agccaaaat 39
<210> 123
<211> 20
<212> DNA
<213> artificial sequence
<400> 123
atatgctccc atctttgacc 20
<210> 124
<211> 39
<212> DNA
<213> artificial sequence
<400> 124
aacggggatg aacggtcaag aaccattgta aacgttacc 39
<210> 125
<211> 36
<212> DNA
<213> artificial sequence
<400> 125
cgcgaacgcc tgggcttggt aaatatagta accaga 36
<210> 126
<211> 48
<212> DNA
<213> artificial sequence
<400> 126
ttcgcattac tgttgggaag ggcaccaatg aaagatattt tgttaaaa 48
<210> 127
<211> 22
<212> DNA
<213> artificial sequence
<400> 127
gagggaaggt aaatattgac gg 22
<210> 128
<211> 22
<212> DNA
<213> artificial sequence
<400> 128
ccctgagagg gttccgatac tc 22
<210> 129
<211> 21
<212> DNA
<213> artificial sequence
<400> 129
atatgtgtcg attataccaa g 21
<210> 130
<211> 49
<212> DNA
<213> artificial sequence
<400> 130
ctcactgccc gctttccagt aaacatagag cttagtttga ctggccttg 49
<210> 131
<211> 48
<212> DNA
<213> artificial sequence
<400> 131
ccaacgtcat agcaccatgg taatttccag tcgatataag actttttc 48
<210> 132
<211> 40
<212> DNA
<213> artificial sequence
<400> 132
agctcatcgc cattccagct ggaagcaact tccgaactgc 40
<210> 133
<211> 20
<212> DNA
<213> artificial sequence
<400> 133
gagatttggc tatttttgag 20
<210> 134
<211> 35
<212> DNA
<213> artificial sequence
<400> 134
gtgtaaagcc tgggtaaatg tcccccattt gaggg 35
<210> 135
<211> 45
<212> DNA
<213> artificial sequence
<400> 135
tggccttctc cagccgccag ggtcgccctg gctacgtgcg ttgcg 45
<210> 136
<211> 20
<212> DNA
<213> artificial sequence
<400> 136
aggtgaaagc tccaattgct 20
<210> 137
<211> 27
<212> DNA
<213> artificial sequence
<400> 137
ccatctgtcg aaagggtact tttgtaa 27
<210> 138
<211> 48
<212> DNA
<213> artificial sequence
<400> 138
aatcaaaaca tcggcattat caccgattga ggttgatggt agttgcag 48
Claims (16)
1. The DNA nano robot is characterized in that the preparation raw materials of the DNA nano robot comprise a combination of staple nucleic acids shown in SEQ ID No. 1-138, a bracket chain nucleic acid solution and a buffer solution;
the molar ratio of the staple nucleic acid to the stent strand nucleic acid in the stent strand nucleic acid solution is 1 (0.05-1.5);
the DNA nano robot is prepared by the following method:
(1) Respectively dissolving the staple nucleic acids of the nucleotide sequences shown in SEQ ID No. 1-138 in water to obtain staple nucleic acid solutions of the nucleotide sequences shown in SEQ ID No. 1-138;
(2) Reacting the staple nucleic acid solution, the bracket chain nucleic acid solution and the buffer solution of the nucleotide sequences shown in SEQ ID No. 1-138 obtained in the step (1) under the condition of cooling to obtain the DNA nano robot;
the cooling method comprises the following steps: maintaining the temperature of the system at 85-95 ℃ for 5-15 min, cooling to 75-85 ℃ for the first time, cooling to 55-65 ℃ for the second time, and cooling to 15-25 ℃ for the third time, thereby completing the cooling.
2. The DNA nanorobot of claim 1, wherein the shape of the DNA nanorobot is C-shaped.
3. The DNA nanorobot of claim 1, wherein the buffer comprises a combination of ethylenediamine tetraacetic acid, magnesium chloride, sodium chloride, and Tris.
4. The DNA nanorobot of claim 3, wherein the molar ratio of ethylenediamine tetraacetic acid to magnesium chloride is 1 (12-13).
5. The DNA nanorobot of claim 3, wherein the molar ratio of ethylenediamine tetraacetic acid to sodium chloride is 1 (12-13).
6. The DNA nanorobot of claim 3, wherein the molar ratio of ethylenediamine tetraacetic acid to sodium chloride is 1 (4 to 6).
7. The DNA nanorobot of claim 3, wherein the pH of Tris is 7 to 8.
8. A method for preparing the DNA nanorobot according to any one of claims 1 to 7, comprising the steps of:
(1) Respectively dissolving the staple nucleic acids of the nucleotide sequences shown in SEQ ID No. 1-138 in water to obtain staple nucleic acid solutions of the nucleotide sequences shown in SEQ ID No. 1-138;
(2) Reacting the staple nucleic acid solution, the bracket chain nucleic acid solution and the buffer solution of the nucleotide sequences shown in SEQ ID No. 1-138 obtained in the step (1) under the condition of cooling to obtain the DNA nano robot;
the cooling method comprises the following steps: maintaining the temperature of the system at 85-95 ℃ for 5-15 min, cooling to 75-85 ℃ for the first time, cooling to 55-65 ℃ for the second time, and cooling to 15-25 ℃ for the third time, thereby completing the cooling.
9. The method according to claim 8, wherein the molar percentage of the staple nucleic acid in each of the staple nucleic acid solutions in the step (1) is 100 to 300nM.
10. The method according to claim 8, wherein the first cooling rate is 5-15 ℃/min.
11. The method according to claim 8, wherein the second cooling rate is 5-15 ℃/min.
12. The method according to claim 8, wherein the third cooling rate is 5-15 ℃/h.
13. The method of claim 8, wherein the cooling is performed under conditions of a metal bath.
14. The preparation method according to claim 8, characterized in that the preparation method comprises the steps of:
(1) Respectively dissolving staple nucleic acids with nucleotide sequences shown in SEQ ID No. 1-138 in water to respectively obtain staple nucleic acid solutions with the mole percentage content of 100-300 nM and the nucleotide sequences shown in SEQ ID No. 1-138;
(2) Reacting the staple nucleic acid solution, the bracket chain nucleic acid solution and the buffer solution of the nucleotide sequences shown in SEQ ID No. 1-138 obtained in the step (1) under the cooling condition of a metal bath to obtain the DNA nano robot; the cooling method comprises the following steps: maintaining the temperature of the system at 85-95 ℃ for 5-15 min, cooling to 75-85 ℃ for the first time, cooling to 55-65 ℃ for the second time, and cooling to 15-25 ℃ for the third time, thereby completing the cooling; the cooling rate of the first cooling is 5-15 ℃/min; the cooling rate of the second cooling is 5-15 ℃/min; the cooling rate of the third cooling is 5-15 ℃/h.
15. Use of a staple nucleic acid in the preparation of a DNA nanorobot, wherein said staple nucleic acid comprises a combination of the staple nucleic acids shown in SEQ ID nos. 1 to 138.
16. Use of a DNA nanorobot according to any one of claims 1 to 7 for the preparation of a medical material or medical device.
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| CN113004362B true CN113004362B (en) | 2023-08-15 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1312723A (en) * | 1998-02-18 | 2001-09-12 | 考丽克萨有限公司 | Compound and method for diagnosis of tuberculosis |
| KR20180108125A (en) * | 2017-03-24 | 2018-10-04 | 서울대학교산학협력단 | Dna structure |
| CN111705299A (en) * | 2019-03-01 | 2020-09-25 | 湖南早晨纳米机器人有限公司 | Preparation method of nano robot and nano robot |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1669450B1 (en) * | 2003-09-30 | 2011-11-09 | AnGes MG, Inc. | Staple type oligonucleotide and drug comprising the same |
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2021
- 2021-02-26 CN CN202110221142.0A patent/CN113004362B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1312723A (en) * | 1998-02-18 | 2001-09-12 | 考丽克萨有限公司 | Compound and method for diagnosis of tuberculosis |
| KR20180108125A (en) * | 2017-03-24 | 2018-10-04 | 서울대학교산학협력단 | Dna structure |
| CN111705299A (en) * | 2019-03-01 | 2020-09-25 | 湖南早晨纳米机器人有限公司 | Preparation method of nano robot and nano robot |
Non-Patent Citations (1)
| Title |
|---|
| Challenges and Perspectives of DNA Nanostructures in Biomedicine;Adrian Keller，et al.;Angew. Chem. Int. Ed.;第59卷(第37期);15818 – 15833 * |
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