CN107881211A - Utilize the method for DNA origami structures structure cascade reaction - Google Patents

Utilize the method for DNA origami structures structure cascade reaction Download PDF

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CN107881211A
CN107881211A CN201711087044.2A CN201711087044A CN107881211A CN 107881211 A CN107881211 A CN 107881211A CN 201711087044 A CN201711087044 A CN 201711087044A CN 107881211 A CN107881211 A CN 107881211A
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dna
enzyme
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foldings
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何丹农
徐艳
陈玮嘉
王萍
金彩虹
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Shanghai National Engineering Research Center for Nanotechnology Co Ltd
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)

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Abstract

The present invention proposes a kind of system that cascade enzyme is built using DNA origami structures, assembled respectively with two kinds of DNA paper foldings assemblings with two kinds of DNA paper foldings with cascade enzyme with DNA assemblings, two kinds of tubulose DNA paper foldings formation, cascade enzyme respectively including cascade enzyme, realize the accurate regulation of enzyme spacing.Tubulose DNA paper folding of the enzyme respectively with the extension of two kinds of different locis will be cascaded be connected, subsequent tubulose DNA paper foldings further hybridize, and so as to the distance between two enzymes that further, reach to regulate and control the purpose of enzyme spacing.Using addressable advantage of paper folding, accuracy controlling cascades the spacing of enzyme.Two kinds of enzymes are respectively assembled in two DNA paper foldings, two enzymes are cascaded in order using the hybridization of paper folding extended chain, improve the efficiency of cascade.

Description

Utilize the method for DNA origami structures structure cascade reaction
Technical field
A kind of method that cascade reaction is built using origami structure of present invention design, and in particular to utilize DNA tubulose paper foldings Structure connects two kinds of cascade enzymes, the complementary sequence hybridization stretched out using paper folding respectively, and then controls the distance between cascade enzyme.This Invention belongs to bio-sensing detection field.
Background technology
Naturally occurring cascade reaction in order ensures that various metabolic activities are efficiently carried out in vivo in organism.It is general and Speech, cascade reaction need at least two enzymes, and the separation of any intermediate product, and new reaction are not needed in continuous catalytic process The addition of substrate.This efficient reactive mode causes the extensive concern of scientific research personnel, and utilizes inorganic metal structure, albumen The various ways such as skeleton, nucleic acid nano structure simulate cascade reaction in vitro.These researchs demonstrate strict control cascade enzyme Spacing, it can effectively change the speed of cascade reaction, the activity for regulation and control cascade enzyme opens new thinking.
In the method for various structure cascade reactions, DNA nanostructure shows its exclusive advantage, wherein DNA paper foldings art With the development for promoting increasingly complex three-dimensional new construction.DNA paper foldings art utilizes hundreds of complementary short ss-DNA（Staple Chain）, under high cation concentration, by the long ss-DNA of 7308 bases（Scaffold chain）It is folded into arbitrary graphic pattern.Pass through Design to staple chain, it can strictly control pattern and the modification of DNA paper foldings；Its good biocompatibility and space can be sought Location property makes its application prospect extensive.Cascade enzyme spacing is controlled in 10 nm or so scope although existing method can be realized, Because spacing is smaller, space steric effect has had a strong impact on packaging efficiency of the cascade enzyme in same paper folding.For this problem, The present invention a kind of system based on tubulose DNA origami structures research cascade enzyme of design, is improved using the advantage of origami structure control The packaging efficiency of enzyme is cascaded, reaches the purpose of control cascade enzyme spacing.
The content of the invention
Regulate and control demand and existing issue for enzymatic activity, present invention aims at：It is anti-using the structure cascade of DNA origami structures The method answered, the system using DNA origami structures structure cascade enzyme is proposed, realizes the accurate regulation of enzyme spacing.
The object of the invention is realized by following proposal：A kind of method that cascade reaction is built using DNA paper foldings, will cascade enzyme The tubulose DNA paper foldings with two kinds of different loci designs are connected respectively, and subsequent tubulose DNA paper foldings further hybridize, including following steps Suddenly：
（1）Enzyme is cascaded to assemble with DNA respectively：Will cascade enzyme respectively with bridging agent 3- (2- pyridines dimercapto) propionic acid N- hydroxysuccinimidyls Imide ester（SPDP）With certain proportion mix 25 DEG C be incubated 2hr after, ultrafiltration washes away excessive SPDP, the two of subsequent SPDP modifications DNA 1, the DNA2 with sulfydryl modification respectively of kind enzyme with the uniform 25 DEG C of overnight incubations of certain proportion, washes away excess using ultrafiltration afterwards DNA, obtain enzyme and DNA complexⅠ and complexⅱ；
（2）Two kinds of tubulose DNA paper foldings are formed：Chain DNA will be replaced to mix with the ratio of equivalent with original DNA staples chain respectively Even, gained mixture is then with certain proportion and single-stranded long chain DNA, if M13 single stranded DNAs are in 1 × TAE-Mg2+Lead in solution system PCR annealing is crossed, forms DNA origami structures, the origami structure of formation washes away excessive staple chain using ultrafiltration, respectively obtains paper folding I and paper folding II；
（3）Enzyme is cascaded to assemble with two kinds of DNA paper foldings respectively：Two kinds cascade enzyme-DNA complexⅠs and complexⅱ respectively with two kinds Tubulose DNA paper foldings I and paper folding II are well mixed with certain proportion, and in 45 DEG C of annealing assemblings, it is multiple to respectively obtain cascade enzyme-paper folding Compound III and compound IV, not connected enzyme and paper folding are separated off by agarose gel electrophoresis, obtain including compound III With the band of compound IV, recovery cascade enzyme-paper folding compound III and compound IV are purified using Filter column；
（4）Two kinds of DNA paper foldings assembling with cascade enzyme：Enzyme-paper folding compound III and compound IV will be cascaded with certain proportion It is well mixed, in 37 DEG C of annealing assemblings, obtain cascading enzyme-paper folding end-product compound V.
By the inventive method, tubulose DNA paper folding of the enzyme respectively with two kinds of different loci designs will be cascaded and be connected, then pipe Shape DNA paper foldings further hybridize, and so as to the distance between two enzymes that further, reach the purpose of regulation and control enzyme spacing.
On the basis of such scheme, described cascade enzyme is the conventional cascade enzyme in this area, and preferably grape is glycoxidative Enzyme（GOx）And horseradish peroxidase（HRP）；Described cascade enzyme and DNA assembling ratio can be the conventional ratio in this area Example, preferable ratio are 1:10.
Described buffer solution is this area routine cushioning liquid, is preferably buffered during enzyme modification DNA molten for pH=7.4,8 mM Na2HPO4、2 mM KH2PO4, 136 mM Na Cl, 2.6 mM K Cl, 1 × PBS, DNA paper foldings synthesis and agarose The buffer solution of gel electrophoresis is pH=8.0,40 mM Tris- acetic acid, 1 mM EDTA, 12.5 mM MgCl2 1×TAE-Mg2+It is molten Liquid.
Described tubulose DNA paper foldings are conventional different size of, preferable a width of 6 nm of tubulose DNA paper foldings in this area, The length of side is 412 nm；Described long-chain single stranded DNA can be the conventional long-chain in this area, preferably M13mp18 single stranded DNAs.
Described preferable paper folding assembling mode comprises the following steps：Single-stranded M13mp18 and the staple chain DNA are mixed Together in 1 × TAE-Mg2+In cushioning liquid, 20 DEG C are annealed to from 90 DEG C in PCR instrument, annealing rate is 0.1 DEG C/10s.
Selected DNA paper foldings sequence can be the conventional sequence in this area, and preferably August in 2010 is published in Nano on 3rd The entitled Programmable Periodicity of Quantum Dot of Letter the 9th the 3367-3372 pages of phases of volume 10 S6- in the Supporting Online Information of Arrays with DNA Origami Nanotubes paper The DNA sequence dna of Table S1 described in S8 pages, each DNA sequence dna are shown in Seq. No1 to 25 DNA；Wherein, with the sequence portions of DNA 1 Divide the sequence for the 3 ' terminal extensions that complementary DNA 3 is the Column 1 of Helix 4；The complementary DNA with the Sequences of DNA 2 4-7 is respectively the sequence of the Column 1/4/7/10 of Helix 43 ' terminal extensions；7-16 points of the sequence DNA replaced in addition Not Wei the Column 5/14/23/32/38/47/56/65/74 of Helix 13 ' terminal extensions sequence；The sequence DNA of replacement 17-25 is respectively the sequence of the Column 6/15/24/33/39/48/57/66/75 of Helix 63 ' terminal extensions, wherein DNA The sequence of 7-16 and DNA 17-25 3 ' terminal extensions is complementary.
Step（3）In, described cascades what enzyme-DNA A complexⅠs and complexⅱ assembled with DNA paper foldings I and paper folding II Mode is the conventional mode in this area, is annealed to 20 DEG C from 45 DEG C preferably in PCR instrument, annealing rate is 0.1 DEG C/15 s。
Further, step（3）In, described cascade enzyme-DNA complexⅠs and complexⅱ and DNA paper foldings I and paper folding II The ratio of assembling is the conventional ratio in this area, and preferable ratio is cascade enzyme-DNA complexⅠs or II：DNA paper foldings I or II= 10:1。
Further, step（3）In, the way of purification of described cascade enzyme-paper folding compound III and compound IV is ability The conventional mode in domain, preferably agarose gel electrophoresis, preferable agarose concentration are 0.5 %, preferable electrophoresis strip Part is the min of 80 V voltage ice baths electrophoresis 80；The way of recycling for cascading enzyme-paper folding compound III and the band of compound IV is ability The conventional mode in domain, is preferably purified using Freeze N Squeeze spin columns.
Step（4）In, the assembling ratio of described cascade enzyme-paper folding compound III and compound IV can be that this area is normal The ratio of rule, preferable ratio are 1:1.Assembling mode can be the conventional assembling mode in this area, preferable using in PCR instrument In 10 DEG C are annealed to from 37 DEG C, annealing rate is 0.1 DEG C/15 s.
The advantage of the invention is that：
（1）Using addressable advantage of paper folding, accuracy controlling cascades the spacing of enzyme.
（2）Two kinds of enzymes are respectively assembled in two DNA paper foldings, using the hybridization of paper folding extended chain by two orderly levels of enzyme Connection, improve the efficiency of cascade.
Embodiment
Technical scheme is further described below by way of specific embodiment.Following embodiment is to this The further explanation of invention, and do not limit the scope of the invention.
Embodiment 1
GOx, HRP assemble with sulfydryl DNA 1 and DNA 2 respectively：100 μl GOx、HRP（2 μM）With SPDP with 1:10 ratios are 1 2 hr are incubated at room temperature in × PBS.30 kD super filter tube centrifuge washings of excessive SPDP（3000g, 10 min）Three times Afterwards, 10 minutes recovery solution of 1000g ultrafiltration, it is ultraviolet quantitative.GOx, HRP of SPDP- modifications respectively with the sulfydryls of 10 times of excess- DNA 1 and DNA 2 is incubated at room temperature overnight in 1 × PBS.Determined by the change for determining light absorption value at 343 nm Measure GOx, HRP and DNA connection ratio.Last unnecessary DNA is removed by the method for centrifugal ultrafiltration（3000g, 10 min, three It is secondary）, 10 minutes recovery solution of 1000g ultrafiltration, obtain GOx-DNA complexⅠs and HRP-DNA complexⅱs.
The paper folding of DNA tubuloses synthesizes：Will replace chain DNA 3, DNA 8-16 and remaining do not replace staple chain equal proportion mix Uniformly, 200 nM are diluted to, are designated as staple combination chain I.Will replace chain DNA 4, DNA 17-25 and remaining do not replace staple Chain equal proportion is well mixed, and is diluted to 200 nM, is designated as staple combination chain II.Take 100 μ l（20 nM）It is single-stranded M13mp18 DNA respectively with 10 μ l staples combination chains I and II 10 10 × TAE-Mg of μ l2+Buffer solution (pH 8), 70 μ l Ultra-pure water is well mixed, assembling of being annealed in PCR.Using 1 × TAE-Mg2+Buffer solution (pH 8) and the μ L of 100 kD 500 The g centrifuge washings of super filter tube 3000 three times, remove unnecessary single stranded DNA, DNA paper foldings I after 10 minutes recovery purifyings of 1000g ultrafiltration With II.
Enzyme is cascaded to assemble with two kinds of DNA paper foldings respectively：By GOx-DNA complexⅠs and HRP-DNA complexⅱs respectively with folding Paper I and II is with concentration ratio 10:1 ratio is in 1 × TAE-Mg2+Mixed in buffer solution, subsequent mixed liquor is in PCR instrument from 45 DEG C 20 DEG C are annealed to, annealing rate is 0.1 DEG C/15 s.Two kinds of obtained PCR mixtures are in 0.5 % Ago-Gels（Using 1 × TAE-Mg2+Buffer solution）After the min of 80 V voltage ice baths electrophoresis 80, rubber tapping is obtained with cascade enzyme-paper folding compound III With the band of compound IV, degumming is further purified using Freeze N Squeeze spin columns, obtain cascade enzyme- Paper folding compound III and compound IV.
Two kinds of DNA paper foldings assembling with cascade enzyme：Cascade enzyme-paper folding compound III and compound are concentrated using super filter tube IV, ultraviolet quantitative concentrations.5 nM are cascaded into the nM compounds IV of enzyme-paper folding compound III and 5 with 1:1 volume ratio is well mixed, 10 DEG C of assemblings are down in 37 DEG C of annealing in PCR, obtain cascade enzyme-paper folding end-product compound V that spacing is 10 nm.
Embodiment 2
GOx, HRP assemble with sulfydryl DNA 1 and DNA 2 respectively：100 μl GOx、HRP（2 μM）With SPDP with 1:10 ratios are 1 2 hr are incubated at room temperature in × PBS.30 kD super filter tube centrifuge washings of excessive SPDP（3000g, 10 min）Three times Afterwards, 10 minutes recovery solution of 1000g ultrafiltration, it is ultraviolet quantitative.GOx, HRP of SPDP- modifications respectively with the sulfydryls of 10 times of excess- DNA 1 and DNA 2 is incubated at room temperature overnight in 1 × PBS.Determined by the change for determining light absorption value at 343 nm Measure GOx, HRP and DNA connection ratio.Last unnecessary DNA is removed by the method for centrifugal ultrafiltration（3000g, 10 min, three It is secondary）, 10 minutes recovery solution of 1000g ultrafiltration, obtain GOx-DNA complexⅠs and HRP-DNA complexⅱs.
The paper folding of DNA tubuloses synthesizes：Will replace chain DNA 3, DNA 8-16 and remaining do not replace staple chain and be well mixed, it is dilute Release to 200 nM, be designated as staple combination chain I.Will replace chain DNA 5, DNA 17-25 and remaining do not replace staple chain mix Uniformly, 200 nM are diluted to, are designated as staple combination chain II.Take 100 μ l（20 nM）Single-stranded M13mp18 DNA respectively with 10 μ l staples combination chains I and II 10 10 × TAE-Mg of μ l2+Buffer solution (pH 8), 70 μ l ultra-pure waters are well mixed, Anneal and assemble in PCR.Using 1 × TAE-Mg2+Buffer solution (pH 8) and the g of 100 kD, 500 μ L super filter tubes 3000 centrifugations are washed Wash and remove unnecessary single stranded DNA three times, DNA paper foldings I and II after 10 minutes recovery purifyings of 1000g ultrafiltration.
Enzyme is cascaded to assemble with two kinds of DNA paper foldings respectively：By GOx-DNA complexⅠs and HRP-DNA complexⅱs respectively with folding Paper I and II is with concentration ratio 10:1 ratio is in 1 × TAE-Mg2+Mixed in buffer solution, subsequent mixed liquor is in PCR instrument from 45 DEG C 20 DEG C are annealed to, annealing rate is 0.1 DEG C/15 s.Two kinds of obtained PCR mixtures are in 0.5 % Ago-Gels（Using 1 × TAE-Mg2+Buffer solution）After the min of 80 V voltage ice baths electrophoresis 80, rubber tapping is obtained with cascade enzyme-paper folding compound III With the band of compound IV, degumming is further purified using Freeze N Squeeze spin columns, obtain cascade enzyme- Paper folding compound III and compound IV.
Two kinds of DNA paper foldings assembling with cascade enzyme：Cascade enzyme-paper folding compound III and compound are concentrated using super filter tube IV, ultraviolet quantitative concentrations.5 nM are cascaded into the nM compounds IV of enzyme-paper folding compound III and 5 with 1:1 volume ratio is well mixed, 10 DEG C of assemblings are down in 37 DEG C of annealing in PCR, are obtained spacing and are cascaded enzyme-paper folding end-product compound V for 18 nm.
Embodiment 3
GOx, HRP assemble with sulfydryl DNA 1 and DNA 2 respectively：100 μl GOx、HRP（2 μM）With SPDP with 1:10 ratios are 1 2 hr are incubated at room temperature in × PBS.30 kD super filter tube centrifuge washings of excessive SPDP（3000g, 10 min）Three times Afterwards, 10 minutes recovery solution of 1000g ultrafiltration, it is ultraviolet quantitative.GOx, HRP of SPDP- modifications respectively with the sulfydryls of 10 times of excess- DNA 1 and DNA 2 is incubated at room temperature overnight in 1 × PBS.Determined by the change for determining light absorption value at 343 nm Measure GOx, HRP and DNA connection ratio.Last unnecessary DNA is removed by the method for centrifugal ultrafiltration（3000g, 10 min, three It is secondary）, 10 minutes recovery solution of 1000g ultrafiltration, obtain GOx-DNA complexⅠs and HRP-DNA complexⅱs.
The paper folding of DNA tubuloses synthesizes：Will replace chain DNA 3, DNA 8-16 and remaining do not replace staple chain and be well mixed, it is dilute Release to 200 nM, be designated as staple combination chain I.Will replace chain DNA 6, DNA 17-25 and remaining do not replace staple chain mix Uniformly, 200 nM are diluted to, are designated as staple combination chain II.Take 100 μ l（20 nM）Single-stranded M13mp18 DNA respectively with 10 μ l staples combination chains I and II 10 10 × TAE-Mg of μ l2+Buffer solution (pH 8), 70 μ l ultra-pure waters are well mixed, Anneal and assemble in PCR.Using 1 × TAE-Mg2+Buffer solution (pH 8) and the g of 100 kD, 500 μ L super filter tubes 3000 centrifugations are washed Wash and remove unnecessary single stranded DNA three times and pass through DNA paper foldings I and II after 10 minutes recovery purifyings of 1000g ultrafiltration.
Enzyme is cascaded to assemble with two kinds of DNA paper foldings respectively：By GOx-DNA complexⅠs and HRP-DNA complexⅱs respectively with folding Paper I and II is with concentration ratio 10:1 ratio is in 1 × TAE-Mg2+Mixed in buffer solution, subsequent mixed liquor is in PCR instrument from 45 DEG C 20 DEG C are annealed to, annealing rate is 0.1 DEG C/15 s.Two kinds of obtained PCR mixtures are in 0.5 % Ago-Gels（Using 1 × TAE-Mg2+Buffer solution）After the min of 80 V voltage ice baths electrophoresis 80, rubber tapping is obtained with cascade enzyme-paper folding compound III With the band of compound IV, degumming is further purified using Freeze N Squeeze spin columns, obtain cascade enzyme- Paper folding compound III and compound IV.
Two kinds of DNA paper foldings assembling with cascade enzyme：Cascade enzyme-paper folding compound III and compound are concentrated using super filter tube IV, ultraviolet quantitative concentrations.5 nM are cascaded into the nM compounds IV of enzyme-paper folding compound III and 5 with 1:1 volume ratio is well mixed, 10 DEG C of assemblings are down in 37 DEG C of annealing in PCR, are obtained spacing and are cascaded enzyme-paper folding end-product compound V for 31 nm.
Embodiment 4
GOx, HRP assemble with sulfydryl DNA 1 and DNA 2 respectively：100 μl GOx、HRP（2 μM）With SPDP with 1:10 ratios are 1 2 hr are incubated at room temperature in × PBS.30 kD super filter tube centrifuge washings of excessive SPDP（3000g, 10 min）Three times Afterwards, 10 minutes recovery solution of 1000g ultrafiltration, it is ultraviolet quantitative.GOx, HRP of SPDP- modifications respectively with the sulfydryls of 10 times of excess- DNA 1 and DNA 2 is incubated at room temperature overnight in 1 × PBS.Determined by the change for determining light absorption value at 343 nm Measure GOx, HRP and DNA connection ratio.Last unnecessary DNA is removed by the method for centrifugal ultrafiltration（3000g, 10 min, three It is secondary）, 10 minutes recovery solution of 1000g ultrafiltration, obtain GOx-DNA complexⅠs and HRP-DNA complexⅱs.
The paper folding of DNA tubuloses synthesizes：Will replace chain DNA 3, DNA 8-16 and remaining do not replace staple chain and be well mixed, it is dilute Release to 200 nM, be designated as staple combination chain I.Will replace chain DNA 7, DNA 17-25 and remaining do not replace staple chain mix Uniformly, 200 nM are diluted to, are designated as staple combination chain II.Take 100 μ l（20 nM）Single-stranded M13mp18 DNA respectively with 10 μ l staples combination chains I and II 10 10 × TAE-Mg of μ l2+Buffer solution (pH 8), 70 μ l ultra-pure waters are well mixed, Anneal and assemble in PCR.Using 1 × TAE-Mg2+Buffer solution (pH 8) and the g of 100 kD, 500 μ L super filter tubes 3000 centrifugations are washed Wash and remove unnecessary single stranded DNA three times and pass through DNA paper foldings I and II after 10 minutes recovery purifyings of 1000g ultrafiltration.
Enzyme is cascaded to assemble with two kinds of DNA paper foldings respectively：By GOx-DNA complexⅠs and HRP-DNA complexⅱs respectively with folding Paper I and II is with concentration ratio 10:1 ratio is in 1 × TAE-Mg2+Mixed in buffer solution, subsequent mixed liquor is in PCR instrument from 45 DEG C 20 DEG C are annealed to, annealing rate is 0.1 DEG C/15 s.Two kinds of obtained PCR mixtures are in 0.5 % Ago-Gels（Using 1 × TAE-Mg2+Buffer solution）After the min of 80 V voltage ice baths electrophoresis 80, rubber tapping is obtained with cascade enzyme-paper folding compound III With the band of compound IV, degumming is further purified using Freeze N Squeeze spin columns, obtain cascade enzyme- Paper folding compound III and compound IV.
Two kinds of DNA paper foldings assembling with cascade enzyme：Cascade enzyme-paper folding compound III and compound are concentrated using super filter tube IV, ultraviolet quantitative concentrations.5 nM are cascaded into the nM compounds IV of enzyme-paper folding compound III and 5 with 1:1 volume ratio is well mixed, 10 DEG C of assemblings are down in 37 DEG C of annealing in PCR, are obtained spacing and are cascaded enzyme-paper folding end-product compound V for 46 nm.
<110>Shanghai National Engineering Research Center for Nanotechnology Co., Ltd
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<213>Artificial sequence
<220>
<400>tacaaaggct atcaaacaac gccaacatgc gcagaggcga attttttttt tttttttttt ttttt
<210>16
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>gaagattgat taagataaga ataaacacat aaatcaatat attttttttt tttttttttt ttttt
<210>17
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>aaggaaaccg aggaccgtcat aaatattcaa ctaatgcaga tatttaaaaa aaaaaaaaaa aaaaa
<210>18
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>agagagataa cccaaaagat taagaggaaa gaactggctc attttaaaaa aaaaaaaaaa aaaaa
<210>19
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>tttaacgtca aaaaaagagg tcatttttcg taacaaagct gctttaaaaa aaaaaaaaaa aaaaa
<210>20
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>cacccagcta caataattct gcgaacgata agggaaccga actttaaaaa aaaaaaaaaa aaaaa
<210>21
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>attctaagaa cgcggggcgc gagctgaatt gtatcatcgc cttttaaaaa aaaaaaaaaa aaaaa
<210>22
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>ttccaagaac gggtcggttg taccaaaact cacattaatt gctttaaaaa aaaaaaaaaa aaaaa
<210>23
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>tgttcagcta atgcaagatt caaaagggag ctcgaattcg tatttaaaaa aaaaaaaaaa aaaaa
<210>24
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>gaatcgccat atttggtcat tgcctgaggg ggatgtgctg catttaaaaa aaaaaaaaaa aaaaa
<210>25
<211>65
<212>DNA
<213>Artificial sequence
<220>
<400>aaataaggcg ttaacaaata tttaaattgc cagctttccg gctttaaaaa aaaaaaaaaa aaaaa

Claims (10)

1. it is a kind of using DNA paper foldings build cascade reaction method, it is characterised in that will cascade enzyme respectively with two kinds of different locis The tubulose DNA paper foldings connection of design, subsequent tubulose DNA paper foldings further hybridize, comprised the steps：
（1）Enzyme is cascaded to assemble with DNA respectively：Will cascade enzyme respectively with bridging agent 3- (2- pyridines dimercapto) propionic acid N- hydroxysuccinimidyls Imide ester（SPDP）With certain proportion mix 25 DEG C be incubated 2hr after, ultrafiltration washes away excessive SPDP, the two of subsequent SPDP modifications DNA 1, the DNA2 with sulfydryl modification respectively of kind enzyme with the uniform 25 DEG C of overnight incubations of certain proportion, washes away excess using ultrafiltration afterwards DNA, obtain enzyme and DNA complexⅠ and complexⅱ；
（2）Two kinds of tubulose DNA paper foldings are formed：Chain DNA will be replaced to mix with the ratio of equivalent with original DNA staples chain respectively Even, gained mixture is then with certain proportion and described long-chain single stranded DNA in 1 × TAE-Mg2+Moved back in solution system by PCR Fire, forms DNA origami structures, and the origami structure of formation washes away excessive staple chain using ultrafiltration, respectively obtains paper folding I and paper folding Ⅱ；
（3）Enzyme is cascaded to assemble with two kinds of DNA paper foldings respectively：Two kinds cascade enzyme-DNA complexⅠs and complexⅱ respectively with two kinds Tubulose DNA paper foldings I and paper folding II are well mixed with certain proportion, and in 45 DEG C of annealing assemblings, it is multiple to respectively obtain cascade enzyme-paper folding Compound III and compound IV, not connected enzyme and paper folding are separated off by agarose gel electrophoresis, obtain including compound III With the band of compound IV, recovery cascade enzyme-paper folding compound III and compound IV are purified using Filter column；
（4）Two kinds of DNA paper foldings assembling with cascade enzyme：Enzyme-paper folding compound III and compound IV will be cascaded with certain proportion It is well mixed, in 37 DEG C of annealing assemblings, obtain cascading enzyme-paper folding end-product compound V.
2. the method for DNA paper foldings structure cascade reaction is utilized according to claim 1, it is characterised in that described cascade enzyme is excellent Choosing for glucose oxidase（GOx）And horseradish peroxidase（HRP）；The assembling ratio of described cascade enzyme and DNA can be with For the conventional ratio in this area, preferable ratio is 1:10.
3. the method for DNA paper foldings structure cascade reaction is utilized according to claim 1, it is characterised in that described buffer solution In, preferably buffer during enzyme modification DNA molten for pH=7.4,8 mM Na2HPO4、2 mM KH2PO4、136 mM Na Cl、2.6 MM K Cl, 1 × PBS；The buffer solution of DNA paper foldings synthesis and agarose gel electrophoresis is pH=8.0,40 mM Tris- Acetic acid, 1 mM EDTA, 12.5 mM MgCl2 1×TAE-Mg2+Solution.
4. the method for DNA paper foldings structure cascade reaction is utilized according to claim 1, it is characterised in that described DNA paper foldings Preferable tubulose a width of 6 nm of DNA paper foldings, the length of side is 412 nm；Described long-chain single stranded DNA is preferably M13mp18 single-stranded DNA。
5. the method for DNA paper foldings structure cascade reaction is utilized according to claim 1, it is characterised in that described preferable folding Paper assembling mode comprises the following steps：Single-stranded M13mp18 and the staple chain DNA are mixed in 1 × TAE-Mg2+Cushioning liquid In, 20 DEG C are annealed to from 90 DEG C in PCR instrument, annealing rate is 0.1 DEG C/10s.
6. the method for DNA paper foldings structure cascade reaction is utilized according to claim 1, it is characterised in that selected DNA paper foldings sequence Row include Seq. No1 to 25 DNA, wherein, it is the Column's 1 of Helix 4 with the complementary DNA 3 of the Sequences of DNA 1 The sequence of 3 ' terminal extensions；Complementary DNA 4-7 are respectively the Column 1/4/7/10 of Helix 4 with the Sequences of DNA 2 The sequence of 3 ' terminal extensions；The sequence DNA 7-16 replaced in addition is respectively the Column 5/14/23/32/38/47/ of Helix 1 The sequence of 56/65/74 3 ' terminal extensions；The sequence DNA 17-25 of replacement is respectively the Column 6/15/24/ of Helix 6 The sequence of 33/39/48/57/66/75 3 ' terminal extensions, wherein DNA 7-16 and the sequence of DNA 17-25 3 ' terminal extensions It is complementary.
7. the method for DNA paper foldings structure cascade reaction is utilized according to claim 1, it is characterised in that described cascade enzyme- The mode that DNA A complexⅠs and complexⅱ assemble with DNA paper foldings I and paper folding II is the conventional mode in this area, preferably 20 DEG C are annealed to from 45 DEG C in PCR instrument, annealing rate is 0.1 DEG C/15 s.
8. the method for DNA paper foldings structure cascade reaction is utilized according to claim 1, it is characterised in that described cascade enzyme- DNA complexⅠs and complexⅱ and the ratio of DNA paper foldings I and paper folding II assembling are the conventional ratio in this area, preferable ratio Example is cascade enzyme-DNA complexⅠs or II：DNA paper foldings I or II=10:1.
9. the method for DNA paper foldings structure cascade reaction is utilized according to claim 1, it is characterised in that described cascade enzyme- The way of purification of paper folding compound III and compound IV is the conventional mode in this area, preferably agarose gel electrophoresis, compared with Good agarose concentration is 0.5 %, and preferable deposition condition is the min of 80 V voltage ice baths electrophoresis 80；Cascade enzyme-paper folding The way of recycling of compound III and the band of compound IV is the conventional mode in this area, preferably using Freeze N Squeeze spin columns are purified.
10. the method for DNA paper foldings structure cascade reaction is utilized according to claim 1, it is characterised in that described cascade enzyme- The assembling ratio of paper folding compound III and compound IV can be the conventional ratio in this area, and preferable ratio is 1:1.Assembling side Formula can be the conventional assembling mode in this area, and preferable using being annealed to 10 DEG C from 37 DEG C in PCR instrument, annealing rate is 0.1 ℃/15 s。
CN201711087044.2A 2017-11-07 2017-11-07 Utilize the method for DNA origami structures structure cascade reaction Pending CN107881211A (en)

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