CN108795939A - Based on DNAzyme DNA paper folding surface aggregates method - Google Patents
Based on DNAzyme DNA paper folding surface aggregates method Download PDFInfo
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- CN108795939A CN108795939A CN201810660560.8A CN201810660560A CN108795939A CN 108795939 A CN108795939 A CN 108795939A CN 201810660560 A CN201810660560 A CN 201810660560A CN 108795939 A CN108795939 A CN 108795939A
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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
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
- C08G73/026—Wholly aromatic polyamines
- C08G73/0266—Polyanilines or derivatives thereof
Abstract
The present invention relates to a kind of based on DNAzyme in the method for DNA paper folding surface aggregates, and make a stretch of the arm chain on the specific site of DNA paper foldings surface, and the G- tetrads with biology catalytic activity are connected in a manner of DNA hybridization.Excessive DNA staples chain, G- tetrads and M13mp18 DNA are with 10:10:1 ratio is uniformly mixed, and PCR annealing is hybridized；In the DNA paper folding mixed liquors for having connected G- tetrads, the reagent needed for G- tetrad catalytic polymerizations is added, controls reaction solution concentration or reaction time etc., you can in original nanostructure, generation of the fixed point catalysis high molecular polymer on single, double chain DNA.Reaction condition is mild, toxicity is low, biological safety is high.The present invention is directed to nanometer materials, and realizing fixed point has the high molecular polymer of excellent conductive capability in DNA paper folding Surface Creations.
Description
Technical field
The present invention relates to a kind of based on DNAzyme in the method for DNA paper folding surface aggregates, and biology is simulated with DNAzyme
Enzymatic activity, in the method that DNA paper foldings surface pinpoints regulation and control catalysed aniline polymerisation.The invention belongs to nano-macromolecule material necks
Domain.
Background technology
Polyaniline is the high molecular polymer for having excellent conductive capability, it has the advantages that synthesize quick, high stability,
And electrical conductive activities can be regulated and controled by simple acid/base chemical doping, therefore be with a wide range of applications.Traditional aniline chemistry closes
It is usually carried out by aoxidizing aniline monomer at method, needs the polarity condition of acid pH, the polyaniline dissolubility of generation is low, limitation
The processing and fabricating in later stage.Therefore people are by using auto-dope instead, improving polyaniline using the methods of template and biocatalyst
Synthesis technology.Wherein biological enzyme has that higher catalytic activity, specificity are strong, activity is controllable, reaction condition is mild
Advantage is a kind of relatively simple and environment amenable synthetic method, can significantly reduce the toxicity and cost when production.Especially
, horseradish peroxidase（HRP）Conducting high polymers object is carried out by application with its efficient catalytic activity and yield
Production.
However usually with the dendritic polyaniline by-product for facing position and contraposition substitution of generation height when HRP catalytic polymerizations
Object, this unordered polymer architecture poorly conductive greatly harm the generation of ordered polymer.In order to improve contraposition polymerization
The production efficiency of polyaniline, polyelectrolyte are used as the template of polyaniline synthesis, and this template can not only provide polyphenyl
Necessity charge compensation when amine synthesizes, and the water solubility of product is improved, it is produced convenient for post-production.DNA is with a large amount of negative
The high molecular polymer of charge is that aniline polymerization reacts ideal template.Especially by hundreds of short chains（Staple chain）With one
Bar long-chain（Scaffold chain）Hybridize the DNA origami structures generated, there is orderly double-spiral structure and a large amount of negative electrical charge, be exhausted
The template of good aniline synthesis.Although largely reducing the generation of branch by-product by the method for templated synthesis,
However the higher catalytic rates of HRP remain difficult to realize that position control enzymatic polymerization reacts on the nanostructure.Thus need to research and develop
A kind of method that the catalysed aniline positioning simultaneous reactions mild condition orderly polymerize.
Invention content
The present invention be directed to existing issue deficiency, propose it is a kind of based on DNAzyme in the side of DNA paper folding surface aggregates
Method.
The present invention is catalyst in G- tetrad of the DNA paper foldings connection with bioenzyme activity, and to contain a large amount of double-strands
DNA origami structure as template, after being adsorbed on DNA paper foldings using aniline, regulation and control high molecular polymer in double-stranded DNA paper folding
The orderly generation on surface, includes the following steps：
（1）G- tetrads are connect with DNA paper foldings：
The chain that makes a stretch of the arm on the specific site of DNA paper foldings surface is designed, is connected with biology catalytic activity in a manner of DNA hybridization
G- tetrads.Excessive DNA staples chain, G- tetrads and M13mp18 DNA are with 10:10:1 ratio is uniformly mixed, PCR
Annealing is hybridized, it is extra it is single-stranded removed through ultrafiltration, ultraviolet quantitative concentrations；
（2）Catalytic polymerization：
In the DNA paper folding mixed liquors for having connected G- tetrads, the reagent needed for G- tetrad catalytic polymerizations, control is added
Reaction solution concentration or reaction time etc., you can in original nanostructure, fixed point catalysis high molecular polymer is in single, double chain DNA
On generation.
The method of the present invention has the advantages that be swift in response, toxicity is low, biological safety is high.
The G- tetrads are carried out at the same time with DNA paper foldings to be connect.
The G- tetrads sequence can be the G- tetrad sequences with class peroxidase activity of this field routine
Row, are published in J. AM. Chem. Soc. 13156-13157 pages of phase entitled A of volume 132 the 38th preferably on July 2nd, 2010
lead(II)-driven DNA molecular device for turn-on fluorescence detection of
It is recorded in page 13156 in the paper of lead (II) ion with high selectivity and sensitivity
DNA sequence dna Seq.No1 is denoted as DNA 1.
The DNA origami structures be triangle, rectangular, strip-form, cube, two dimension or three dimensional DNA nanostructure, more
Good origami structure is triangle DNA paper foldings, and the best length of side is 130 nm.
Selected DNA paper foldings sequence is the sequence of this field routine, is published in J. AM. within preferably on 2 17th, 2010
Chem. the entitled Gold nanoparticle self-similar of Soc. the 10th the 3248-3249 pages of phases of volume 132
The Supporting Online Infromation of the paper of chain structure organized by DNA origami
In S11-S16 pages recorded in slave A01 to Loo DNA sequence dna, the replacement chain DNA 2-16 of stretching be sequence A61, A49,
The base of A04, A20, A30, B61, B49, B04, B20, B30, C61, C49, C04, C20, C30,5 ' terminal extensions, preferably
DNA sequence dna is shown in DNA2-16 sequence tables Seq.No2-16.
The catalytic polymerization can be biological enzyme arylamine class, phenols, diamine class generation high molecular polymer
Polymerisation, preferably aniline polymerization are reacted.
The polymerisation buffer solution is this field popular response solution, preferably 4.5 1 × phosphate-buffereds of pH
Liquid（1×PBS）, a concentration of 10 mM Na2HPO4, 1.76 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl.
The concentration of substrate of the biological enzyme is in 1-4 mM.
The catalytic polymerization time is in 10-180 min.
The advantage of the invention is that：
（1）G- tetrad of the modification with class bioenzyme activity in DNA paper foldings utilizes DNAzyme catalytic polymerizations, reaction
Mild condition, toxicity are low, biological safety is high.
（2）The present invention is directed to nanometer materials, and realize fixed point has excellent conductive capability in DNA paper folding Surface Creations
High molecular polymer.
Description of the drawings
Fig. 1 is the atomic force microscope phenogram after 1 polymerisation of case study on implementation.
Specific implementation mode
Technical scheme of the present invention is further described below by way of specific embodiment.Embodiment below is to this
The further explanation of invention, and do not limit the scope of the invention.
Embodiment 1
DNA paper foldings are assembled with G- tetrads：By DNA 1-2 and remaining do not replace staple chain and M13mp18 DNA（20 nM）
10:1 ratio is uniformly mixed PCR annealing, using 1 × TAE-Mg2+500 μ L super filter tubes of buffer solution (pH 8) and 100 kD
Centrifuge washing is three times（3000 g are centrifuged, 1000 g recycling）It washes away extra single stranded DNA to pass through, and 1000g ultrafiltration is returned for 10 minutes
DNA paper foldings after purification are received, paper folding and G- tetrad nanocrystal compositions are quantified with ultraviolet-uisible spectrophotometer（It is denoted as composite I）It is dense
Degree.
Polyaniline generates：10 mM aniline solutions use vinegar acid for adjusting pH to 4.5 first.10 nM composite Is, 2 mM aniline, 2
mM H2O2It is uniformly mixed and is incubated 10min, be denoted as reaction solution I.0.1 mM hemins are added in reaction solution I（Hemin）Keep aniline poly-
It closes on DNA paper foldings surface, reacts 10 min.Sample drop is added in mica surface after taking 3 μ L reactions, 3 min is adsorbed, using ultra-pure water
Cleaning, N2Mica surface is dried up, sample is scanned using tapping-mode under gas phase condition.
Fig. 1 is the atomic force microscope phenogram after 1 polymerisation of case study on implementation, characterizes aniline and occurs on paper folding surface
Product after polymerisation, from the figure, it can be seen that white bright spot characterizes generation of the polyaniline in the site.
Embodiment 2
DNA paper foldings are assembled with G- tetrads：By DNA 1-11 and remaining do not replace staple chain and M13mp18 DNA（20 nM）
10:1 ratio is uniformly mixed PCR annealing, using 1 × TAE-Mg2+500 μ L super filter tubes of buffer solution (pH 8) and 100 kD
Centrifuge washing is three times（3000 g are centrifuged, 1000 g recycling）It washes away extra single stranded DNA to pass through, and 1000g ultrafiltration is returned for 10 minutes
DNA paper foldings after purification are received, paper folding and G- tetrad nanocrystal compositions are quantified with ultraviolet-uisible spectrophotometer（It is denoted as composite I）It is dense
Degree.
Polyaniline generates：10 mM aniline solutions use vinegar acid for adjusting pH to 4.5 first.10 nM composite Is, 3 mM aniline, 2
mM H2O2It is uniformly mixed and is incubated 10min, be denoted as reaction solution I.0.1 mM hemins are added in reaction solution I（Hemin）Keep aniline poly-
It closes on DNA paper foldings surface, reacts 60 min.
Embodiment 3
DNA paper foldings are assembled with G- tetrads：By DNA 1-16 and remaining do not replace staple chain and M13mp18 DNA（20 nM）
10:1 ratio is uniformly mixed PCR annealing, using 1 × TAE-Mg2+500 μ L super filter tubes of buffer solution (pH 8) and 100 kD
Centrifuge washing is three times（3000 g are centrifuged, 1000 g recycling）It washes away extra single stranded DNA to pass through, and 1000g ultrafiltration is returned for 10 minutes
DNA paper foldings after purification are received, paper folding and G- tetrad nanocrystal compositions are quantified with ultraviolet-uisible spectrophotometer（It is denoted as composite I）It is dense
Degree.
Polyaniline generates：10 mM aniline solutions use vinegar acid for adjusting pH to 4.5 first.10 nM composite Is, 4 mM aniline, 2
mM H2O2It is uniformly mixed and is incubated 10min, be denoted as reaction solution I.0.1 mM hemins are added in reaction solution I（Hemin）Keep aniline poly-
It closes on DNA paper foldings surface, reacts 60 min.
Embodiment 4
DNA paper foldings are assembled with G- tetrads：By DNA 1-16 and remaining do not replace staple chain and M13mp18 DNA（20 nM）
10:1 ratio is uniformly mixed PCR annealing, using 1 × TAE-Mg2+500 μ L super filter tubes of buffer solution (pH 8) and 100 kD
Centrifuge washing is three times（3000 g are centrifuged, 1000 g recycling）It washes away extra single stranded DNA to pass through, and 1000g ultrafiltration is returned for 10 minutes
DNA paper foldings after purification are received, paper folding and G- tetrad nanocrystal compositions are quantified with ultraviolet-uisible spectrophotometer（It is denoted as composite I）It is dense
Degree.
Polyaniline generates：10 mM aniline solutions use vinegar acid for adjusting pH to 4.5 first.10 nM composite Is, 2 mM aniline, 2
mM H2O2It is uniformly mixed and is incubated 10min, be denoted as reaction solution I.0.1 mM hemins are added in reaction solution I（Hemin）Keep aniline poly-
It closes on DNA paper foldings surface, reacts 120 min.
Embodiment 5
DNA paper foldings are assembled with G- tetrads：By DNA 1-16 and remaining do not replace staple chain and M13mp18 DNA（20 nM）
10:1 ratio is uniformly mixed PCR annealing, using 1 × TAE-Mg2+500 μ L super filter tubes of buffer solution (pH 8) and 100 kD
Centrifuge washing is three times（3000 g are centrifuged, 1000 g recycling）It washes away extra single stranded DNA to pass through, and 1000g ultrafiltration is returned for 10 minutes
DNA paper foldings after purification are received, paper folding and G- tetrad nanocrystal compositions are quantified with ultraviolet-uisible spectrophotometer（It is denoted as composite I）It is dense
Degree.
Polyaniline generates：10 mM aniline solutions use vinegar acid for adjusting pH to 4.5 first.10 nM composite Is, 1 mM aniline, 2
mM H2O2It is uniformly mixed and is incubated 10min, be denoted as reaction solution I.0.1 mM hemins are added in reaction solution I（Hemin）Keep aniline poly-
It closes on DNA paper foldings surface, reacts 180 min.
<110>Shanghai National Engineering Research Center for Nanotechnology Co., Ltd
<120>A method of based on DNAzyme in DNA paper folding surface aggregates
<160>16
<210>1
<211>43
<212>DNA
<213>Artificial sequence
<400>
aataataata ataataataa taattttggg tgggtgggtg ggt
<210>2
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<213>Artificial sequence
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attattatta ttattattat tatttttgcg cctgttattc taagagcgcg attccagagc ctaattt
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attattatta ttattattat tatttttagc atgtatttca tcgtaggaat caaacgattt tttgttt
<210>4
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<213>Artificial sequence
<400>
attattatta ttattattat tatttttatg gtttatgtca caatcaatag atattaaac
<210>5
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<213>Artificial sequence
<400>
attattatta ttattattat tatttttttg acggaaatac atacataaag ggcgctaata tcagaga
<210>6
<211>67
<212>DNA
<213>Artificial sequence
<400>
attattatta ttattattat tatttttgag ccagcgaata cccaaaagaa catgaaatag caatagc
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<213>Artificial sequence
<400>
attattatta ttattattat tatttttgcg cctgttattc taagaacgcg attccagagc ctaattt
<210>8
<211>67
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<213>Artificial sequence
<400>
attattatta ttattattat tatttttagc atgtatttca tcgtaggaat caaacgattt tttgttt
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<213>Artificial sequence
<400>
attattatta ttattattat tatttttttc gagctaagac ttcaaatatc gggaacgag
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attattatta ttattattat tattttttaa ttgctttacc ctgactatta tgacgcatag taagagc
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attattatta ttattattat tatttttgtt tgcgtcacgc tggtttgccc caagggagcc cccgatt
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attattatta ttattattat tatttttcga ccagtacatt ggcagattca cctgattgc
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attattatta ttattattat tatttttgaa tacgtaacag gaaaaacgct cctaaacagg aggccga
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<213>Artificial sequence
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attattatta ttattattat tattttttaa aacattagaa gaactcaaac tttttataat cagtgag
Claims (9)
1. it is a kind of based on DNAzyme DNA paper folding surface aggregates method, which is characterized in that DNA paper foldings connection have life
The G- tetrads of object enzymatic activity, and using contain a large amount of double-strands DNA origami structure as template, be adsorbed on DNA using aniline
After paper folding, regulation and control high molecular polymer includes the following steps in the orderly generation on DNA paper foldings surface：
（1）G- tetrads are connect with DNA paper foldings：
The chain that makes a stretch of the arm on the specific site of DNA paper foldings surface is designed, is connected with biology catalytic activity in a manner of DNA hybridization
G- tetrads.Excessive DNA staples chain, G- tetrads and M13mp18 DNA are with 10:10:1 ratio is uniformly mixed, PCR
Annealing is hybridized, it is extra it is single-stranded removed through ultrafiltration, ultraviolet quantitative concentrations；
（2）Catalytic polymerization：
In the DNA paper folding mixed liquors for having connected G- tetrads, the reagent needed for G- tetrad catalytic polymerizations, control is added
Reaction solution concentration or reaction time, you can in original nanostructure, fixed point catalysis high molecular polymer is on single, double chain DNA
Generation.
2. according to claim 1 based on DNAzyme DNA paper folding surface aggregates method, it is characterised in that it is described
G- tetrads are carried out at the same time with DNA paper foldings and connect.
3. according to claim 1 based on DNAzyme DNA paper folding surface aggregates method, it is characterised in that it is described
G- tetrad sequences are preferably Seq.No1.
4. according to claim 1 based on DNAzyme DNA paper folding surface aggregates method, it is characterised in that it is described
DNA origami structures are triangle, rectangular, strip-form, cube, two dimension or three dimensional DNA nanostructure, and more preferably origami structure is
Triangle DNA paper foldings, the best length of side are 130 nm.
5. according to claim 1 based on DNAzyme DNA paper folding surface aggregates method, it is characterised in that selected DNA
Paper folding sequence is preferably sequence table Seq.No2-16.
6. according to claim 1 based on DNAzyme DNA paper folding surface aggregates method, it is characterised in that it is described
Catalytic polymerization can generate the polymerisation of high molecular polymer for biological enzyme arylamine class, phenols, diamine class, preferably
It is reacted for aniline polymerization.
7. according to claim 1 based on DNAzyme DNA paper folding surface aggregates method, it is characterised in that it is described
Polymerisation buffer solution is this field popular response solution, preferably 4.5 1 × phosphate buffers of pH（1×PBS）, dense
Degree is 10 mM Na2HPO4, 1.76 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl.
8. according to claim 1 based on DNAzyme DNA paper folding surface aggregates method, it is characterised in that it is described
The concentration of substrate of biological enzyme is in 1-4 mM.
9. according to claim 1 based on DNAzyme DNA paper folding surface aggregates method, it is characterised in that it is described
The catalytic polymerization time is in 10-180 min.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109652480A (en) * | 2018-12-28 | 2019-04-19 | 上海纳米技术及应用国家工程研究中心有限公司 | A method of constructing collection pencil DNA nanostructure |
| CN110862065A (en) * | 2019-11-27 | 2020-03-06 | 厦门大学 | Nano electronic component manufactured by using structural DNA as template and method thereof |
| CN112143727A (en) * | 2020-09-03 | 2020-12-29 | 华中科技大学 | DNA origami structure, method for closing and releasing same and use as drug delivery system |
| CN114249891A (en) * | 2021-08-23 | 2022-03-29 | 南京大学深圳研究院 | Method for polymerizing dopamine monomer on DNA origami by light control |
| US11513076B2 (en) | 2016-06-15 | 2022-11-29 | Ludwig-Maximilians-Universität München | Single molecule detection or quantification using DNA nanotechnology |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN110862065A (en) * | 2019-11-27 | 2020-03-06 | 厦门大学 | Nano electronic component manufactured by using structural DNA as template and method thereof |
| CN112143727A (en) * | 2020-09-03 | 2020-12-29 | 华中科技大学 | DNA origami structure, method for closing and releasing same and use as drug delivery system |
| CN114249891A (en) * | 2021-08-23 | 2022-03-29 | 南京大学深圳研究院 | Method for polymerizing dopamine monomer on DNA origami by light control |
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Application publication date: 20181113 |