CN110643604B - Tweezer-shaped composite nano probe and preparation method and application thereof - Google Patents

Tweezer-shaped composite nano probe and preparation method and application thereof Download PDF

Info

Publication number
CN110643604B
CN110643604B CN201910848227.4A CN201910848227A CN110643604B CN 110643604 B CN110643604 B CN 110643604B CN 201910848227 A CN201910848227 A CN 201910848227A CN 110643604 B CN110643604 B CN 110643604B
Authority
CN
China
Prior art keywords
dna
artificial sequence
solution
gold nanorods
composite nano
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910848227.4A
Other languages
Chinese (zh)
Other versions
CN110643604A (en
Inventor
丁宝全
刘沣嵩
蒋乔
刘清
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Center for Nanosccience and Technology China
Original Assignee
National Center for Nanosccience and Technology China
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Center for Nanosccience and Technology China filed Critical National Center for Nanosccience and Technology China
Priority to CN201910848227.4A priority Critical patent/CN110643604B/en
Publication of CN110643604A publication Critical patent/CN110643604A/en
Application granted granted Critical
Publication of CN110643604B publication Critical patent/CN110643604B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
    • G01N2021/3155—Measuring in two spectral ranges, e.g. UV and visible

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a tweezer-shaped composite nano probe and a preparation method and application thereof. The composite nano probe is a composite nano probe structure formed by inducing self-assembly of rod-shaped gold nano particles by taking a DNA origami nano structure in a tweezers shape as a template. The composite nano probe can identify a specific substrate, namely reduced glutathione. The composite nano probe has the advantages of simple and convenient operation, high sensitivity, mild detection conditions and the like when detecting the reduced glutathione. Theoretically, the composite nano probe can enter cytoplasm by means of endocytosis of cells, and the concentration of reduced glutathione in the cells can be detected on the premise of not destroying the biological activity of the cells. The invention has wide application prospect in detecting the substrate concentration at the cell level.

Description

Tweezer-shaped composite nano probe and preparation method and application thereof
Technical Field
The invention belongs to the field of nano science, and particularly relates to a tweezers-shaped composite nano probe, and a preparation method and application thereof.
Background
When the size of a substance is reduced from the macroscopic scale to the nanometer scale, many physicochemical properties of the substance are significantly changed. For example, many noble metal particles range in size from 10 to 100 nanometers because they are smaller than the mean free path of electrons in the metal solid; and the energy band of the metal is gradually separated into densely arranged energy levels, and the conductive property, the optical property and the like of the metal are changed. One of the major changes is: electrons in the nanoparticles of the noble metal are collected on the surface of the metal particles to generate surface plasmon (surface plasmon). The surface plasmons cause the surface of the metal nanoparticles to carry an electric charge. When an external light source is incident, the surface plasmon absorbs energy of a specific wavelength of light to generate collective vibration. When the frequency of the incident light is close to the natural vibration frequency of the surface plasmon of the metal particle, the absorption value of the light reaches the maximum and the surface plasmon resonance effect occurs.
When the surface plasmon resonates, a strong electromagnetic field is significantly generated around the metal particles. The strength and distribution of the electromagnetic field can be solved by maxwell's system of equations. When one metal particle spatially self-assembles with other metal particles, the electromagnetic field of the surface plasmon can be significantly enhanced. Therefore, it is necessary to find a method for precisely assembling metal nanoparticles.
The DNA paper folding technology provides a feasible idea for accurately assembling the metal nano particles. The DNA origami is a nano-structure assembled by a long-chain circular DNA single chain extracted from M13 bacteriophage and a plurality of artificially designed and synthesized short-chain DNAs through strict base complementary pairing principle. The method has the advantages that the shape of the DNA origami can be designed artificially and the accurate position on the DNA origami can be positioned. The DNA origami is used as a template for assembling the metal nano-particles, and the spatial position and the included angle of the metal nano-particles can be freely regulated and controlled. This provides powerful and critical technical support for the study of the phenomenon of metal surface plasmon resonance.
Glutathione is a small molecule polypeptide widely present in organisms. The catalyst is divided into a reduction type and an oxidation type. The reduced glutathione is formed by connecting one molecule of glutamic acid, one molecule of cysteine and one molecule of glycine by virtue of peptide bonds. Because cysteine residues in the glutathione contain sulfydryl with reducibility, the glutathione has stronger reducibility. In organism cells, glutathione has important functions of participating in enzyme catalytic reaction, maintaining protein activity, eliminating excessive oxygen free radicals and the like. Therefore, detection of reduced glutathione is of great interest to understand the biological effects at the cellular level.
The existing method for detecting glutathione is usually based on chemical reaction or ultraviolet spectrophotometry, and has a plurality of problems, such as high detection limit, need to destroy biological tissues before detecting biological samples, complicated detection method and the like.
Disclosure of Invention
The invention aims to provide a tweezer-shaped composite nanoprobe and a preparation method and application thereof.
In order to achieve the object of the present invention, the tweezer-shaped composite nanoprobe provided by the present invention is a composite nanoprobe structure formed by inducing self-assembly of rod-shaped gold nanoparticles (Au nanoparticles) using a tweezer-shaped DNA origami nanostructure as a template.
In a first aspect, the present invention provides a method for preparing a pincerlike composite nanoprobe, comprising the steps of:
A. synthesizing a gold nanorod, and modifying the surface of the gold nanorod with a specific sequence DNA with a mercapto functional group;
B. synthesizing a DNA origami with a specific capture chain at a pre-designed site;
C. assembling the gold nanorods modified in the step A and DNA origami to obtain a tweezers-shaped composite nanoprobe;
the specific sequence DNA with the sulfhydryl functional group in the step A is as follows: 5 '-TATTATTATTATTATTTTT-SH-3' and 5 '-TTTTTTTTTTTTTTTAGCG-SH-3' (SEQ ID NO: 174-175);
the DNA origami in the step B is assembled by 1M 13mp18 bacteriophage long-chain DNA and 172 short-chain DNA through base complementary pairing, wherein the 172 short-chain comprises 32 capture chains; the nucleic acid sequences of the 172 short-chain DNAs and the long-chain DNAs are respectively shown as SEQ ID NO. 1-173;
and C, hybridizing the modified gold nanorods with the capture chains on the DNA origami through the specific sequence DNA with the mercapto functional groups modified on the surfaces of the gold nanorods, and assembling to obtain the tweezers-shaped composite nanoprobe.
In the foregoing method, step a includes:
a1, synthesizing seed crystal solution;
a2, growing gold nanorods by using the seed crystals synthesized in the step A1 as a substrate;
a3, purifying the gold nanorods obtained in the step A2;
a4, modifying the DNA with a special sequence of a sulfhydryl functional group on the surface of the gold nanorod.
Step a1 includes: adding 50ul of 2 mass percent chloroauric acid solution into 9.5ml CTAB solution, stirring uniformly, adding 1ml of 6mM sodium borohydride solution under the stirring condition, and carrying out water bath at 30 ℃ for 6-12h to obtain a seed crystal solution.
Step a2 includes: 100ml of CTAB solution was added to the reactor, 780ul of 2% chloroauric acid solution was then added with stirring, 700ul of 10mM silver nitrate solution and 480ul of 100mM ascorbic acid solution were added with stirring, and then 160ul of the seed crystal solution prepared in step A1 was added, stirred uniformly (stirring for 2min), and water bath was carried out at 30 ℃ for 12 hours to obtain a colloidal solution containing gold nanorods.
Step a3 includes: and D, centrifuging the colloidal solution obtained in the step A2 at 3000rpm for 20min, and removing the precipitate to obtain the purified gold nanorods.
Step a4 includes:
(1) adding water into artificially synthesized DNA with a specific sequence of a sulfhydryl functional group to prepare 100uM DNA solutions respectively, and adding TCEP to a final concentration of 20mM to ensure that all sulfhydryl groups are in a reduction state;
(2) mixing 800ul of 5 XTBE buffer solution, 40ul of 1% SDS solution and 400ul of 5M NaCl solution, adding water to dilute the mixture to a total volume of 4ml, and adjusting the pH value to 3.6 by hydrochloric acid to obtain a modification solution;
wherein, the composition of the 1 XTBE buffer solution is as follows: 89mM Tris, 89mM boric acid and 2mM EDTA, pH8.0, in water;
(3) under the condition of shaking, adding 100ul of the 100uM solution obtained in the step (1) into the modification solution, adding the gold nanorods purified in the step A3 under the condition of shaking, and carrying out water bath at 30 ℃ for 6h to obtain modified gold nanorods; wherein, the mol ratio of the gold nanorods to each DNA is 1:2500 respectively.
In the foregoing method, step B includes:
b1, artificially synthesizing M13mp18 phage long-chain DNA and 172 short-chain DNA, mixing the long-chain DNA and the short-chain DNA according to the molar ratio of 1: 5-1: 10, and adding Mg into the mixture2+Self-assembly is carried out in the 1 XTAE buffer solution;
the self-assembly conditions were: the PCR amplificator was set up with the following program: cooling from 95 deg.C to 65 deg.C, and maintaining each gradient for 5min at every 5 deg.C; cooling from 65 deg.C to 25 deg.C, and maintaining each gradient for 10min at each temperature of 1 deg.C; the whole annealing process is about 8 hours;
b2, after the self-assembly is finished, the self-assembly product and Mg are contained2+Mixing the 1 XTAE buffer solution according to the volume ratio of 1:4, adding the mixture into a centrifugal column with the molecular weight cutoff of 100KDa, and centrifuging the mixture for 3min at 5000 rpm; centrifuging to remove excessive short-chain DNA;
b3 adding the solution into a centrifugal columnMg2+Repeatedly centrifuging and washing the 1 XTAE buffer solution for 3 times to obtain the DNA origami.
Wherein said Mg is contained2+The composition of the 1 XTAE buffer solution is: 0.04M Tris, 0.02M acetic acid, 0.003M EDTA and 0.0125M magnesium acetate, pH 8.0.
In the foregoing method, step C includes:
c1, mixing the modified gold nanorods with the DNA origami according to the mol ratio of 4: 1-10: 1 (preferably 8:1) (keeping the concentration of the DNA origami at 3nM), and adding Mg into the mixture2+Self-assembly is carried out in the 1 XTAE buffer solution;
the self-assembly conditions were: the PCR amplificator was set up with the following program: cooling from 45 deg.C to 25 deg.C, and maintaining each gradient for 5min at every 1 deg.C; when the temperature is kept at 25 ℃ for 5min, the temperature is raised to 45 ℃ for the next cycle, the reaction is finished for 6 cycles, and the reaction is stored at 4 ℃;
c2, after the self-assembly is finished, carrying out 0.5% -2% agarose gel electrophoresis on the self-assembly product, wherein the electrophoresis conditions are as follows: 85V, 1 h;
c3, cutting gel and recovering an electrophoresis product, namely the tweezer-shaped composite nano probe.
In step C2, the solution used to prepare the gel was 10mM MgCl 21 × TBE buffer solution. The solution used for electrophoresis was the same as that used for the gel preparation described above.
In a second aspect, the present invention provides a tweezer-like composite nanoprobe prepared according to the method.
The composite nano probe can identify a specific substrate, namely reduced glutathione. When the composite nano probe identifies a substrate, the angles of two cantilevers of a tweezers structure of the composite nano probe can be changed in an opening and closing mode.
After the composite nano probe is contacted with a substrate and subjected to angle change, the circular dichroism spectrum of the composite nano probe changes at a wavelength of 400-900 nanometers.
The optical signal change and the two-arm switch of the composite nano probe are as follows: when the two arms of the composite nanoprobe are opened, the circular dichromatic optical signal is weakened; when the two arms of the composite nano probe are closed, the circular dichroism optical signal is enhanced.
When reduced glutathione is introduced, glutathione cleaves the disulfide bonds between the composite nanoprobes, causing the arms of the forceps to open.
The length of the gold nanorods on the composite nano probe is 40 +/-4 nm, and the diameter of the gold nanorods on the composite nano probe is 10 +/-1 nm; the absorption peak of the ultraviolet spectrum is in the wavelength region of 760 + -20 nm.
And detecting the wavelength range of the composite nano probe by circular dichroism spectrum within 400-900 nm.
In a third aspect, the invention provides an application of the composite nano probe in detection of reduced glutathione.
In the actual detection process, the reduced glutathione solution is added into the composite nano probe solution and incubated for 6-12h at room temperature (25 ℃ -30 ℃). The spectral signal is detected in the circular dichroism spectrum, and the concentration of the reduced glutathione can be calculated according to the weakening of the spectral signal. The linear detection range of the reduced glutathione is 0.1 mM-5 mM, and the lower detection limit is 0.1 mM.
Preferably, the reduced glutathione and the composite nano-probe contain 10mM MgCl 21 × TBE buffer solution.
Preferably, the wavelength range of the circular dichroism spectrum detection composite nano probe is 400-900 nm.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the composite nano probe structure provided by the invention is assembled by a gold nanorod and a DNA origami structure. The structural form of the DNA origami can be artificially controlled, and all the sites for assembling the capture chains of the gold nanorods are also accurately controllable. The assembling form of the gold nanorods can be controlled by adjusting the site of the capture chain, so that the strength of the initial circular dichroism spectrum signal of the nanoprobe is controlled. And the signal range of the circular dichroism spectrum of the composite nano probe is controlled by controlling the length-diameter ratio of the gold nanorods.
And secondly, in view of the fact that the reaction of cutting the disulfide bond by the reduced glutathione is very reliable, the process of opening the disulfide bond is amplified through the change of the circular dichroism optical signal caused by the structural opening and closing of the composite nano probe, so that the method has the advantages of high response sensitivity, few samples required for detection and the like. Meanwhile, compared with a chemical oxidation-reduction method, the composite nano probe provided by the invention has the advantages of mild and controllable reaction conditions, non-toxic and harmless reaction products and the like.
Thirdly, the composite nano probe structure provided by the invention is assembled by a gold nanorod and a DNA origami structure; the gold nanorods have stable chemical properties and no biotoxicity, and the DNA paper folding structure is formed by assembling DNA chains according to a base complementary pairing rule, so that the gold nanorods have good biocompatibility. The size of the composite nano probe provided by the invention is in a nano level; theoretically, the glutathione can be taken up by cells through endocytosis and other ways, so that the method has the potential possibility of detecting the reduced glutathione at the cellular level and has wide application prospect in the field of biomedicine.
And (IV) the invention takes the DNA origami nano structure in the shape of tweezers as a template, induces the two gold nanorods to assemble a certain included angle, and changes the included angle between the two gold nanorods through the interaction between the substrate and the DNA origami structure. The change in angle between the rods can be detected by the difference in circular dichroism signals. Compared with the traditional detection technology, the composite nano probe provided by the invention has the advantages of low cost, convenient and quick detection means, low detection limit and the like.
Drawings
Fig. 1 is a schematic diagram of a three-dimensional model of a composite nanoprobe material prepared in example 3 of the present invention in an open or closed state before and after introduction of reduced glutathione.
Fig. 2A and 2B are design diagrams of the composite nanoprobe of the present invention.
FIG. 3 is an electron microscopic representation of the gold nanorods constituting the composite nanoprobe in example 2 of the present invention.
FIG. 4 is a band of the composite nanoprobe structure of example 3 according to the present invention, which is generated by agarose gel purification. Among them, lanes 1-6 are self-assembly product samples.
FIG. 5 is a TEM image of the closed state (A) and the open state (B) of the composite nanoprobe in example 4 of the present invention.
FIG. 6 is a graph showing the change of circular dichroism spectrum signals of the composite nanoprobe before and after glutathione detection in example 4 of the present invention.
Detailed Description
The invention provides a composite nano probe for detecting the concentration of reduced glutathione based on circular dichroism spectrum.
According to the invention, the glutathione and the area containing the disulfide bond in the composite nano probe are subjected to oxidation-reduction reaction, so that the appearance of the composite nano probe is changed from a closed state to an open state; the open-close change of the morphology of the composite nanoprobe can be shown in the circular dichroism spectrum. When the reduced glutathione is not added, the composite nano probe structure is in a closed state, and the circular dichroism spectrum signal is strong; after the reduced glutathione is added, the disulfide bonds in the structure are opened, the composite nano probe is in an opened state, and the circular dichroism signal is weakened.
The invention adopts the following technical scheme:
in one aspect, the invention provides a tweezers-shaped composite nano probe formed by assembling gold nanorods and DNA origami structures.
In the composite nano probe structure, the composite nano probe is a composite nano structure formed by assembling a DNA origami structure and a gold nanorod.
The preparation method of the composite nano probe structure provided by the invention comprises the following steps:
(1) synthesizing the gold nanorods by using a seed crystal growth method, and modifying the surfaces of the gold nanorods by using specific sequence DNA with sulfhydryl functional groups.
(2) Synthesizing a DNA origami nano structure with a specific capture chain at a pre-designed site.
(3) And assembling the gold nanorods and the DNA origami nano structure to obtain the composite nano probe structure.
(4) The resulting composite nanostructure was purified by agarose gel electrophoresis.
Preferably, the synthesis of gold nanorods in step (1) is synthesized by a seed method, and the required seed is gold nanocluster consisting of several tens of gold atoms, dispersed in 100mM cetyltrimethylammonium bromide (CTAB) solution.
Preferably, the length of the gold nanorods synthesized in the step (1) is 40 +/-4 nm, and the diameter of the gold nanorods is 10 +/-1 nm; the absorption peak of the ultraviolet spectrum is in the wavelength region of 760 + -20 nm.
Preferably, the gold nanorods synthesized in step (1) are dispersed in a cetyltrimethylammonium bromide (CTAB) solution with a concentration of 100 mM.
Preferably, the DNA strand sequence modified on the gold rod in step (1) is as follows (SEQ ID NO: 174-175):
S10：TATTATTATTATTATTTTT-SH
S15：TTTTTTTTTTTTTTTAGCG-SH
preferably, in the step (1), the molar ratio of two DNA strands used for modification to the gold nanorods is 2500:1 respectively.
Preferably, in step (1), the gold nanorods are modified with DNA strands, which should be carried out in a special modification solution. The preparation method of the modifying liquid comprises the following steps: 800ul of 5 XTBE buffer solution, 40ul of 1% SDS solution and 400ul of 5M NaCl solution were mixed, diluted with water to a total volume of 4ml, and the pH was adjusted to 3.6 with hydrochloric acid.
Preferably, when the gold nanorods are modified with DNA in step (1), the DNA strands are mixed with the gold nanorods and then incubated in a thermostatic water bath at 30 ℃ for 6h to complete the modification.
Preferably, the DNA origami structure of step (2) is 172 artificially designed short-chain DNAs (comprising 32 capture chains); and a natural DNA long chain which is extracted from the M13mp18 bacteriophage and has the length of 7249 basic groups, and the nucleic acid nano structure is assembled by strict basic group complementary pairing. A DNA long strand of 7249 bases in length was extracted from the M13mp18 phage. The nucleic acid sequences of the 172 short-chain DNAs and the long-chain DNAs are respectively shown in SEQ ID NO. 1-173.
Preferably, the 172 short chains in step (2) are designed with the aid of software cadnano. Examples of such short chains are as follows:
Staple1：TAAAGTACCATTCATCTCATTACTCCATGTTACTTAGCAG
Staple2：CAATACGCTGAGAGCCAGGATAGAAAAACATAGTTAATTGC AATAA
Staple3：TTTATCACCGGAACAA
Staple4：CACCAATAATAATTTTTTAAACAACGAACTGGGAACCTG
Staple5：GGCATTTTCAAATGAACGCCATGGGCGCATCGTAACGC
preferably, the DNA origami structure is designed with 32 capturing chains in total, 32 corresponding short chains are replaced, and the sequences of the 32 capturing chains are respectively shown in SEQ ID NO. 1-32.
Preferably, the above 32 capture strands located on the DNA origami nanostructure are complementary to DNA strands modified on the surface of the gold nanorods, thereby achieving the purpose of assembling the gold rods.
Preferably, in step (2), the mole ratio of the long chains extracted from M13mp18 phage added in the assembly of DNA origami structures to the artificially designed short chains is 1: 10.
Preferably, in step (2), the extracted long chain is present in the M13mp18 phage at a concentration of 10 nM.
Preferably, in step (2), the DNA origami nanostructure is assembled in the presence of Mg 2+1 XTAE in buffer solution.
Preferably, in the step (2), the assembled DNA origami nanostructure is annealed in a PCR amplification instrument to control the reaction temperature change, the annealing temperature program is from 95 ℃ to 65 ℃, each gradient is performed at 5 ℃, and the retention time of each gradient is 5 min; from 65 ℃ to 25 ℃, each temperature gradient is a gradient at 1 ℃, and the retention time of each temperature gradient is 10 min; the whole annealing process is about 8 h.
Preferably, in step (2), the assembled DNA origami nanostructure requires purification to remove excess short-chain DNA. The purification steps are as follows: mixing the obtained annealed product with Mg2+Mixed in a 1:4 volume ratio and added to a 100kDa spin column for centrifugation.
Preferably, in the step (3), the gold nanorods are modified by DNA strands with thiol functional groups, and the DNA strands modified on the gold nanorods are hybridized with capture strands extending out of the DNA origami structure in a base complementary pairing manner to assemble the composite nanostructure.
Preferably, in the step (3), the modified gold nanorods and the DNA origami nanostructure with the capture strand are mixed in a molar ratio of 8: 1. And (3) in a PCR amplification instrument, staying for 5 minutes at 45-25 ℃ every 1 ℃, and after staying for 5 minutes at 25 ℃, heating to 45 ℃ for the next cycle, wherein the cycle is 6, and the assembly is finished.
Preferably, in the step (4), the agarose gel used for purifying the composite nano probe structure has a mass fraction of 1%, and the solution for preparing the gel contains 10mM MgCl 21 × TBE buffer solution.
Preferably, in the step (4), the voltage for electrophoresis is 85V, and the electrophoresis time is 1 h. The solution environment required for electrophoresis was the same as that described above for the agarose gel preparation.
The invention carries out a large number of experiments, and regulates the space position of the gold nanorod assembly by changing the capture chain site on the DNA origami nanostructure; and a series of gold nanorods with ultraviolet-visible light absorption wavelength from 700nm to 790nm are used as raw materials, and the relation between the size, distance and mutual included angle of the gold nanorods and the response of circular dichroism optical signals is researched.
In another aspect, the present invention provides a system based on the interaction of composite nanoprobe materials with reduced glutathione, which can change the morphology and cause the optical response of the composite nanoprobe structure to the circular dichroism spectrum to change. Therefore, the signal change of the structure in different forms in a circular dichroism spectrum after the reaction with the reduced glutathione and the composite nano probe structure is utilized to detect the concentration of the reduced glutathione.
Preferably, the reduced glutathione and the composite nano-probe contain 10mM MgCl 21 × TBE buffer solution.
Preferably, the reaction temperature of the reduced glutathione with the composite nanoprobe is 25 ℃.
Preferably, the reaction time of the reduced glutathione and the composite nano probe is 6-12 h.
Preferably, the wavelength range of the circular dichroism spectrum detection composite nano probe is 400-900 nm.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The instruments and reagents used in the invention are as follows:
the instrument comprises the following steps:
MastercycLer Pro gradient PCR instrument (Eppendorf company, germany); UV-2600 type UV-visible spectrophotometer (shimadzu corporation, japan); Milli-Q Reference ultrapure water treatment system (Merck Millipore, Germany); FE20K pH meter (mettler Toledo, switzerland); 5424 Small high speed centrifuge (Eppendorf Co., Germany); 5810R Large high speed centrifuge (Eppendorf Co., Germany); vortex Genius model 3 oscillating mixer (IKA, germany); model 7700 transmission electron microscope (Hitachi corporation, japan); JY-SCZ9 type vertical gel electrophoresis apparatus (Jun-Oriental Co., China); YP5002 electronic analytical balance (Mettler Toledo, switzerland).
Reagent:
the preparation of the composite nano structure related in the invention mainly uses the following reagents:
magnesium chloride, Tris base, boric acid, EDTANA2Glacial acetic acid, sodium chloride, Sodium Dodecyl Sulfate (SDS), cetyldimethylammonium bromide (CTAB), chloroauric acid trihydrate, sodium borohydride, ascorbic acid, silver nitrate, agarose (agarose). The reagents used above were all analytical grade, purchased from Sigma-Aldrich.
Buffer solution used:
containing Mg 2+1 XTAE buffer solution (1 XTAE/Mg)2+Buffer solution) was made up of: 0.04M Tris, 0.02M acetic acid, 0.003M EDTA and 0.0125M magnesium acetate, pH 8.0.
The composition of the 1 × TBE buffer solution was: 89mM Tris, 89mM boric acid and 2mM EDTA, pH8.0, in water.
Example 1 preparation and purification of DNA origami nanostructures with Capture sites
The template strand (native long strand extracted from M3mp18 phage, SEQ ID NO:173) and helper fold strand (artificially designed short strand DNA, SEQ ID NO:33-172), capture strand (short strand DNA designed for assembly of gold nanorods, SEQ ID NO:1-32) were first mixed in a molar ratio of 1:10: 10.
The number and the sites of the capturing chains can be adjusted according to specific requirements, so that the purpose of controlling the assembling form of the gold nanorods is achieved.
At 1 XTAE/Mg2+Annealing under the condition of buffer solution; the annealing conditions are as follows: from 95 ℃ to 65 ℃, each 5 ℃ is a gradient, and the retention time of each gradient is 5 min; from 65 ℃ to 25 ℃, each temperature gradient is a gradient at 1 ℃, and the retention time of each temperature gradient is 10 min; the whole annealing process is about 8 hours; after annealing, the DNA origami structure sample is added into a centrifugal column (the cut-off molecular weight is 100KDa), and 1 XTAE/Mg is added2+Centrifuging the buffer solution to remove excessive short-chain DNA; centrifuging at 5000rpm for 3min, and repeating for three times.
Example 2 preparation, purification and modification of gold nanorods
The preparation steps of the gold nanorods are as follows:
and (1) synthesizing a seed crystal solution.
And (2) growing the gold nanorods by using the seed crystals synthesized in the step (1) as a substrate.
And (3) purifying the gold nanorods obtained in the step (2) by using a centrifugal machine.
And (4) modifying the surface of the gold nanorod with single-stranded DNA with sulfydryl.
The reagent used for synthesizing the seed crystal in the step (1) is as follows: a chloroauric acid solution with the mass fraction of 2%, a sodium borohydride solution with the mass fraction of 6mM and a CTAB solution with the mass fraction of 100 mM.
50ul of 2% chloroauric acid solution was added to 9.5ml CTAB solution and stirred until uniform. 6mM of sodium borohydride solution prepared by low-temperature water and 1ml of sodium borohydride solution are rapidly added under strong stirring, and the seed crystal is synthesized in water bath at 30 ℃ for 6 hours.
The reagents used in step (2) are: a 2% by mass chloroauric acid solution, a 10mM silver nitrate solution, a 100mM ascorbic acid solution, and a 100mM CTAB solution.
Firstly, adding 100ml of CTAB solution into a reaction vessel, then adding 780ul of 2% chloroauric acid solution under stirring, adding 700ul of 10mM silver nitrate solution under stirring, adding 480ul of 100mM ascorbic acid solution under stirring, adding 160ul of the seed crystal solution prepared in the step (1), stirring for 2min to uniformly disperse, and then putting the solution into a 30 ℃ water bath for 12h to obtain the prepared gold nanorod.
The gold nanorods prepared in the step (2) often contain some impurities, such as gold nanospheres, which need to be removed in a centrifugal mode.
And (3) putting the gold nanorod colloid prepared in the step (2) into a centrifuge, centrifuging at the rotating speed of 3000rpm for 20min, wherein the lower-layer precipitate is impurities such as gold nanospheres, and removing the lower-layer impurities to obtain the purified gold nanorod colloid (shown in figure 3).
The synthetic thiol-bearing DNA strand was dissolved in 100uM with ultrapure water and TCEP was added to a final concentration of 20mM to ensure that all thiol groups were in a reduced state.
The DNA strand sequence with a thiol group is as follows (SEQ ID NO: 174-175):
S10：TATTATTATTATTATTTTT-SH
S15：TTTTTTTTTTTTTTTAGCG-SH
800ul of 5 XTBE solution, 40ul of 1% SDS solution and 400ul of 5M NaCl solution were mixed, diluted with ultrapure water to a total volume of 4ml, and the pH was adjusted to 3.6 with hydrochloric acid to obtain a modification solution.
Under the condition of shaking, 100ul of reduced 100uM S10 or S5DNA solution is respectively added into the modification solution, and then the gold nanorods purified in the step (3) are added under the condition of shaking. The amount of gold nanorods added was controlled to be 1/2500 (molar ratio) of S10 or S15DNA strand. And finally transferring the modification solution to a water bath at 30 ℃ for incubation for 6h to complete modification.
Example 3 preparation of tweezers-like composite nanoprobes
The DNA origami nanostructure with capture strands prepared in example 1 was mixed with the purified gold nanorods of example 2 in a molar ratio of 1:8, and the concentration of the DNA origami was maintained at 3 nM. And (3) uniformly mixing, putting into a PCR amplification instrument, setting the temperature program to be 45-25 ℃, staying for 5min at each temperature, when the temperature stays for 5min at 25 ℃, heating to 45 ℃ for the next cycle, reacting for 6 cycles, and storing at 4 ℃ to finish the assembly of the composite nano probe structure. However, the gold nanorods are excessively arranged in the assembled structure and need to be removed by means of gel electrophoresis.
With a solution containing 10mM MgCl21% agarose gel with mass percent of 1% is prepared by using the 1 XTBE buffer solution. The mixed solution of the composite nanoprobe structure and the excessive gold nanorods, which are reacted in the PCR amplification instrument, is added with 1/10 volumes of 50% glycerol as the loading buffer solution of gel electrophoresis.
The sample was electrophoresed for 1h with 85V voltage, and it was observed that the mixed solution of the electrophoresed composite nanoprobe structure and the excess gold nanorods was divided into two bands in the agarose gel, as shown in fig. 4, the band with faster electrophoresis speed was the gold nanorods, and the band with slower electrophoresis speed was the composite nanoprobe structure. And finally, cutting and recovering the strips belonging to the composite nano probe structure to obtain the tweezers-shaped composite nano probe.
Fig. 1 is a schematic diagram of a three-dimensional model of the composite nanoprobe material prepared in this example in an open or closed state before and after introduction of reduced glutathione. In the figure, the composite nano probe structure is formed by assembling a tweezers-shaped DNA origami structure and two gold nanorods. The DNA origami nanometer structure in the shape of tweezers consists of two cantilevers for combining gold nanorods and a middle hinge containing a disulfide bond. Reduced glutathione in the substrate to be detected can cut off disulfide bonds on the middle hinge of the composite nano probe, so that the composite nano probe structure can be opened from closed state.
FIGS. 2A and 2B are design drawings of the composite nanoprobe of the present invention (design software: cadnano).
Example 4 response of composite nanoprobes to reduced glutathione
Reduced glutathione was added to the composite nanoprobe constructed in example 3 at a certain concentration and kept at a constant temperature of 30 ℃ for 6 hours. Reduced glutathione will react with the closed composite nanoprobe structure (fig. 5, a); the reacted composite nanoprobes were in the open state (fig. 5, B). The signal change of the composite nano probe structure after the reaction with the reduced glutathione is represented by a circular dichroism spectrometer in the wavelength range of 400 nm-900 nm, and the content of the reduced glutathione in the detected sample can be calculated by calculating the signal intensity shown in figure 6. The linear detection range of the reduced glutathione is 0.1 mM-5 mM, and the lower detection limit is 0.1 mM.
According to the invention, through self-assembly of the DNA origami nano structure and the gold nanorod, a composite nano probe structure is constructed, and the structure can be changed from a closed state to an open state in the environment of reduced glutathione by introducing a disulfide bond functional group into a specific position. The open-close change of the structural morphology of the composite nano probe can be accurately reflected in an optical signal of the circular dichroism spectrometer.
The composite nano probe structure provided by the invention can be used for detecting the concentration of reduced glutathione in a substrate, and compared with the traditional detection method, the composite nano probe structure has the advantages of mild and controllable reaction conditions, non-toxic and harmless reaction products, good biocompatibility and the like, and has great application value in the aspects of analytical chemistry, biological detection, intracellular environment monitoring and the like.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> national center for Nano science
<120> tweezers-shaped composite nano probe and preparation method and application thereof
<130> KHP191114445.0
<160> 175
<170> SIPOSequenceListing 1.0
<210> 1
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaaaaaaaaa aaaaactttt taaattagta aattaccagt tatgggagcg gata 54
<210> 2
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aaaaaaaaaa aaaaaggcgt gtaaagcctg ggggagacgg ccataaagtc agaacgattc 60
a 61
<210> 3
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aaaaaaaaaa aaaaacagga tacgagccgg aagcccttca cagttcagta atttggtaaa 60
g 61
<210> 4
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ataataataa taatagacta acaaaggagc aaatccttga cccttctata acat 54
<210> 5
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ataataataa taatacgcct tgccatcttt tcactcagag cagccattta acatccatat 60
a 61
<210> 6
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aaaaaaaaaa aaaaacttca ttgcgttgcg ctcgtttgcg atgaacggct atcaagctat 60
t 61
<210> 7
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ataataataa taatatattc tttagcgtca gacaggaggt cctgaattat tttctcaaat 60
a 61
<210> 8
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ataataataa taatattagg agaattcaaa catttaatta agaatacgca atac 54
<210> 9
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aaaaaaaaaa aaaaacgaat gagtgagcta actgtggttt ttggagc 47
<210> 10
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ataataataa taatacaaaa gcgctaaatt attcttagat ttctggcgaa gaac 54
<210> 11
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ataataataa taataaaaat acagagtttg aatatatatg tttaatgccg agta 54
<210> 12
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ataataataa taataaaaaa atcaccggaa ccagccaccc tccaaatatt ctacattctg 60
c 61
<210> 13
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aaaaaaaaaa aaaaaacgat ttttgtcagt gaacgaacta aagttttgaa tagg 54
<210> 14
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
aaaaaaaaaa aaaaaggaat ttgttaacga gaagaaaaat cgttagtggt tgatata 57
<210> 15
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ataataataa taataaaggc gttttcatcg gcaagagccg cgagcgtaaa ttaattttaa 60
a 61
<210> 16
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ataataataa taatatacaa cagggataat tactgcttct atttttgtca cgcaaatta 59
<210> 17
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
aaaaaaaaaa aaaaatgcat tttaacaacg taattacagg catagttcag gagg 54
<210> 18
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
aaaaaaaaaa aaaaagccgg aagattttta attattttaa tttcaaccgg ggtt 54
<210> 19
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aaaaaaaaaa aaaaaaaggg ctttccagtc ggggccaacg agcatgtaaa tggt 54
<210> 20
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ataataataa taataattct catagccccc ttagccgcca atttgccagg caagacggtg 60
t 61
<210> 21
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ataataataa taataaagcc aatgaagaga aacacct 37
<210> 22
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
aaaaaaaaaa aaaaaacaat caacatatag gcttaac 37
<210> 23
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
aaaaaaaaaa aaaaaataga tcaaaaacgg atatagttga gtagcattcc tcaga 55
<210> 24
<211> 62
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
aaaaaaaaaa aaaaaatcga tcgtaatcat ggtccgaaaa tgatagcgaa aagaagagca 60
ac 62
<210> 25
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ataataataa taatataccc ccaatactga ttgaaat 37
<210> 26
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ataataataa taataacgca cccacattac aaagagtcaa cgaccagttg ctgg 54
<210> 27
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ataataataa taatacacct agcgacagaa tcacgattgg tgcaccctac cgcgaaagaa 60
c 61
<210> 28
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
aaaaaaaaaa aaaaaaatgg ccttcctaag agtacattca acagtacaaa ccgccacc 58
<210> 29
<211> 62
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
aaaaaaaaaa aaaaaaccaa tttcctgtgt gaaaacgctg gatcgtcaaa aatagcgttt 60
ac 62
<210> 30
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ataataataa taatatttgc gatagcagca ccgaacaaat aatcaagaat cagaatgcaa 60
a 61
<210> 31
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ataataataa taataaatca aacgtcacca atggccagaa gcgggagccg gtattataac 60
t 61
<210> 32
<211> 64
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
aaaaaaaaaa aaaaaagttg ccgctcacaa ttccgagagt tatccccctc gagccagtaa 60
ataa 64
<210> 33
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
catttggcag atattaccgc cagc 24
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
acccgtcgga ttctccgtga gg 22
<210> 35
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
cacttgcctg agtacaacag acagcaaagt attagacttt acttc 45
<210> 36
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
attttgacgc agttgaaa 18
<210> 37
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
taattctaac attacaaagc tgtgtcgaaa tccgcaac 38
<210> 38
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
cctaagaggc gatatcagat tttattgagg caagtttta 39
<210> 39
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
caccaataat aattttttaa acaacgaact gggaacctg 39
<210> 40
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cttttgctcc aatagctttc aacggcggat tgaccgtg 38
<210> 41
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
tttagtaccg ccacccacag ataaacaggg aacgagggta gcgac 45
<210> 42
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ggctcgaggt gaatttctca gcccttagaa aggacaaaaa ggcaga 46
<210> 43
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
cggtgtacag actttgaaag aggacaga 28
<210> 44
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
cattgcaatg gatttctggt cttaggagca ctaacaag 38
<210> 45
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
cgaagccctt tttaagaaac aa 22
<210> 46
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gaatcatagc tacaagagat aaataataac ggaataat 38
<210> 47
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
taatatccag aacaattcac catatcaaag attagagccg tcttt 45
<210> 48
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
agtttgacca t 11
<210> 49
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ggcttatgtt ttgacaagaa aagatagccg aacaacca 38
<210> 50
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cagacgaaat accaatcttg agaggcgcag acggtgtc 38
<210> 51
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
ggcaacaaaa tagcgcataa ataaaccacc acatttcgg 39
<210> 52
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
atcatacagt tacatagacg gcaaacgtag aaaatcag 38
<210> 53
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
agaagtttaa tgaaaatagg att 23
<210> 54
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tattatatca aaaacattaa acgacagtat cggcctgg 38
<210> 55
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
acccgacaat gacaacaatc gtcacctaat gctaaccctc gagagg 46
<210> 56
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
actatcaaga tacaggctga caagggaacc gaactaaa 38
<210> 57
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tattttagcg atagcatttc ataatggaag ggttattt 38
<210> 58
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
caagagtcca cctgattgca taaagattaa gttgggtaac cg 42
<210> 59
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
ctaccagaac aacagttaat gccc 24
<210> 60
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
aattagaggt gaggcggtag acaatgtaaa tctttcattc caataa 46
<210> 61
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
aactcgcggg gacaggaacg gt 22
<210> 62
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
aaacaagtta atatgatcgc actccagccc t 31
<210> 63
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
ggcgcgagct g 11
<210> 64
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
cctaaaacac t 11
<210> 65
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
ttctttgatt agtagacctg acagtgcctc gacaactcgt attcc 45
<210> 66
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ccggttgata a 11
<210> 67
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
aatcagtgag gccacgcgaa ccgaacgatc attttgcgga acaaa 45
<210> 68
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
tttcctccct ctgagactcc tcaagaga 28
<210> 69
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
tgaatttacc gttccagtaa gccatt 26
<210> 70
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
gggcgaaaaa ccgcgccagg cacattagct attacgccag ctca 44
<210> 71
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
tcaaactatc ggcctaataa acatcaccaa tacatttgag gagaa 45
<210> 72
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
acgccagaat tgaatcgaaa cctggctgcg caactgttgg t 41
<210> 73
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
tgtttaacgt g 11
<210> 74
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
ttcagtcgtg ccagtc 16
<210> 75
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
ggaacaacta aagggcggag taattaccca tttggtagat acat 44
<210> 76
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
gcctgagagt cttataaatt aatgccggtt gg 32
<210> 77
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
ctggaaggtc gctacaagaa aattcatcaa tataataa 38
<210> 78
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
attgaggaag gttatctaag gt 22
<210> 79
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
cctgcctcct cagattagcg taaagacaaa agggcatg 38
<210> 80
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
atatgtaaga gactaataac gtgcgtagat tttcaaat 38
<210> 81
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
gcgagaactg agaaatcgcg cccatatcaa aattaaat 38
<210> 82
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
ggattatgaa aacggctcat ttctgccagt ttgagcaa 38
<210> 83
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
ctcagagcca cctgagttcc atcgcgcttt tgcgggatcc aa 42
<210> 84
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
gcaagcaatt agttagttaa gagaaggaaa ccgagtat 38
<210> 85
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
gcgaacctct agctatct 18
<210> 86
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
atccaacacc gcctgcaaaa gcgtaatttt ccctaaagtg caaaga 46
<210> 87
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
tttgagactc attatgggct tccagcgatt ataccaaa 38
<210> 88
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
aaaaaaccat ggaattagag ccagagt 27
<210> 89
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
tacgaaaatc tccaaaaatt ttctgcagga cgagagggtg gtcatt 46
<210> 90
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
ggcatcaaag aaaccagcct tgaaacgcaa agacacca 38
<210> 91
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
ggcattttca aatgaacgcc atgggcgcat cgtaacgc 38
<210> 92
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
taaagtacca ttcatctcat tactccatgt tacttagcag 40
<210> 93
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
aggatagctg gcttgcatgc ctgcgga 27
<210> 94
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
tagcaaggcc ggaccagtag caccatta 28
<210> 95
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
accgttgtag tggcaccagt atttttgccc gaacgttgat 40
<210> 96
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
ttgggttatc taagaacgcg agg 23
<210> 97
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
agtagcaatt atttcataaa ataaaggtgg caacatag 38
<210> 98
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
gccattaaaa atactgatag ccagtacata gatttaaaag gt 42
<210> 99
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
tccaatcttt atcactttga agtaaaacag aaataaac 38
<210> 100
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
attaaccacc agcagaagat tagtctgagt gaattcccat aatagt 46
<210> 101
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
ctgaggcttg cagggagttg ac 22
<210> 102
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
agatttgctg aacctcaaag tcacatagtg aagcaagacc ccaata 46
<210> 103
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
acagtacctt ttcgtcagat gaatatac 28
<210> 104
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
aaaaatggaa a 11
<210> 105
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
ttttagagag gcgactgccc gcgatcggtg cgggcag 37
<210> 106
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
atcgtattgg gtctatcata aaggga 26
<210> 107
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
agcctttctt accagtaatt gagaactggc atgatggg 38
<210> 108
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
ttgctcagta ccagttttgc tcacgttgta atgccactac gattg 45
<210> 109
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
tccgaaatcg gcaaaatccc ttcagc 26
<210> 110
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
gtgtactggt aataggtcag aagtttgcca ttaaaggtga atgaa 45
<210> 111
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
ggccccacgc ataaccgaca tgtacaaaag gaat 34
<210> 112
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
caaccaggcg ctaaatgtta gactgcctgt ttg 33
<210> 113
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
accgttcaat aaaataaggc ttgtatcatc gcctggac 38
<210> 114
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
caatacgctg agagccagga tagaaaaaca tagttaattg caataa 46
<210> 115
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
ataggaacct atattcgg 18
<210> 116
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
acccgagatg ggtataagta a 21
<210> 117
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
gagaatagac g 11
<210> 118
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
tttagctata ttttttatgc gtcaacttta aaacactcat ctagg 45
<210> 119
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
agaatagccc ggcggtcctt gttattaaaa cgacggccag ta 42
<210> 120
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
cgcgaacgag tgtaaacgag a 21
<210> 121
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
tgtatcaccg tactagcgta attgctttac agaggctttg agata 45
<210> 122
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
caatatctac aaaggtaatc gcaaatatcc ggcaccgctt ctggg 45
<210> 123
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
acagttgata acctatttaa ccctgattat cagatatt 38
<210> 124
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
aacctattat tctgcgccac ctaatcaatt catatggttt acaca 45
<210> 125
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
ccgagctcga atctctagag gatccccg 28
<210> 126
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
gaaacaccag aaacaaagta caacgaa 27
<210> 127
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
gttgttcgcc ctgaacacaa cgttttccca gtcacgtg 38
<210> 128
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
gaaccaagaa ctaattcgtc a 21
<210> 129
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
ttgagcagca aagatagggt tgagt 25
<210> 130
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
gactccacac cagttgccta aagggggatg tgctgggg 38
<210> 131
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
atgagcaaca gtattaaaga acgtg 25
<210> 132
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
tcatcttcat cgtagccact gcggatttgc ccataaatca aa 42
<210> 133
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
attagcactt tccacaccct gccttattac gcagtgac 38
<210> 134
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
gatttgccct gaaattcgtc aggtcttctt ttacgtcaaa 40
<210> 135
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
agtatagccc ggtcgtctta attgtgactt tttcatgagg ga 42
<210> 136
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
aggattagga ttagagtttc aaattgcgaa cctaaaacga aagag 45
<210> 137
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
tgaaattacc ttcagaggac gctaaccgcc agttgagta 39
<210> 138
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
accgccaccc tcagaactac attgcgcctc agcagcgaaa gaccg 45
<210> 139
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
aaagagtctg tccaaatggc tataaaactt aaaagtttga gttca 45
<210> 140
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
acggggtcag tgcccattga ctgtagcgta aatattgacg gaacc 45
<210> 141
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
ctgcccaaat ccaataggct ttaaaccgcc tgcagtttgg aa 42
<210> 142
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
gtttccaaaa ggagcctttt ccagactacg tttagctgac cctgac 46
<210> 143
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
agtgccgtcg agagaaatga aaaaggctcc attaaacggg taaag 45
<210> 144
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
gcactaccaa gagaattggc tatttccttg attacagga 39
<210> 145
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
catccttgat accgatagac gcctgattta ggcgtaagag aata 44
<210> 146
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
taagaggcag agccgagcca ctttgtcaca atcaatat 38
<210> 147
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
acagtgcccg tatacaccac cttttcggaa ccgattgagg gataa 45
<210> 148
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
agaatgaaaa atctaaagag ggacataaga cgaactttta tcgtag 46
<210> 149
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
tgatgatgaa aaactgaaga gc 22
<210> 150
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
gcccgtaaca caccctcatt ttcagggata gc 32
<210> 151
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
taataccctc aatcaataat ttacaaggtc tgaatgctgt atagaa 46
<210> 152
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
tatgcaactt agaaagaaga ttgtttggat tatacaaa 38
<210> 153
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
gcatagtaag ttttgccaga gggaaatgtt gagcgagt 38
<210> 154
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
atctagttgg caaatcaact caatctaacc tcc 33
<210> 155
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
gaacgagtaa atcatacctt tgagcggaat tatcaaac 38
<210> 156
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
tttatcaccg gaacaa 16
<210> 157
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
ttaaacatcg gatagcaacc gactttggaa agc 33
<210> 158
<211> 11
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
tcagaaatgt c 11
<210> 159
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
aaacctaaat atcgtctggg ataggtcacg ttggac 36
<210> 160
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
ttcgccaatc ataaaacagg aaaccaggca aagcgc 36
<210> 161
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
tacactaatc atagcc 16
<210> 162
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
taaaatcggt ttatcagccg atctaacgga acgtccagag caaagc 46
<210> 163
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
ccaatgtacc gcattaacct gagaagtgtt tttat 35
<210> 164
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
tattaatttc aagaataaat cccaatcaga acaaacatga aagtat 46
<210> 165
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
tttgtctgaa acaggaaaaa cgctcatgga aa 32
<210> 166
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
aaatgattcg cataagagag ccttaaaatc ctgtcatac 39
<210> 167
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
aattgctcat ttaaatcaag a 21
<210> 168
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
atggcttttg atgaattcac ataatcaggt caccgacttg agagt 45
<210> 169
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
catcttctga cctas 60
satttaacaa cgccaac 33
<210> 170
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
gcatcaaaaa gattaas 60
sgtagctcaa catgt 33
<210> 171
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
acgacaataa acaacas 60
sacaagcaag ccgtt 33
<210> 172
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
cagagcataa agcts 60
stcaccatca atatgat 33
<210> 173
<211> 7249
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
aatgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60
atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120
cgttcgcaga attgggaatc aactgttata tggaatgaaa cttccagaca ccgtacttta 180
gttgcatatt taaaacatgt tgagctacag cattatattc agcaattaag ctctaagcca 240
tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg 300
ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag 360
tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt 420
cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca 480
tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540
aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt 600
ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt 660
aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg 720
atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780
tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840
caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt 900
ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960
aatatccggt tcttgtcaag attactcttg atgaaggtca gccagcctat gcgcctggtc 1020
tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc 1080
gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140
caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200
caaagatgag tgttttagtg tattcttttg cctctttcgt tttaggttgg tgccttcgta 1260
gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320
caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1380
cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1440
tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500
attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560
ttttggagat tttcaacgtg aaaaaattat tattcgcaat tcctttagtt gttcctttct 1620
attctcactc cgctgaaact gttgaaagtt gtttagcaaa atcccataca gaaaattcat 1680
ttactaacgt ctggaaagac gacaaaactt tagatcgtta cgctaactat gagggctgtc 1740
tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga aactcagtgt tacggtacat 1800
gggttcctat tgggcttgct atccctgaaa atgagggtgg tggctctgag ggtggcggtt 1860
ctgagggtgg cggttctgag ggtggcggta ctaaacctcc tgagtacggt gatacaccta 1920
ttccgggcta tacttatatc aaccctctcg acggcactta tccgcctggt actgagcaaa 1980
accccgctaa tcctaatcct tctcttgagg agtctcagcc tcttaatact ttcatgtttc 2040
agaataatag gttccgaaat aggcaggggg cattaactgt ttatacgggc actgttactc 2100
aaggcactga ccccgttaaa acttattacc agtacactcc tgtatcatca aaagccatgt 2160
atgacgctta ctggaacggt aaattcagag actgcgcttt ccattctggc tttaatgagg 2220
atttatttgt ttgtgaatat caaggccaat cgtctgacct gcctcaacct cctgtcaatg 2280
ctggcggcgg ctctggtggt ggttctggtg gcggctctga gggtggtggc tctgagggtg 2340
gcggttctga gggtggcggc tctgagggag gcggttccgg tggtggctct ggttccggtg 2400
attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg 2460
aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg 2520
ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg 2580
gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt 2640
taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt 2700
ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat 2760
tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt 2820
ttgctaacat actgcgtaat aaggagtctt aatcatgcca gttcttttgg gtattccgtt 2880
attattgcgt ttcctcggtt tccttctggt aactttgttc ggctatctgc ttacttttct 2940
taaaaagggc ttcggtaaga tagctattgc tatttcattg tttcttgctc ttattattgg 3000
gcttaactca attcttgtgg gttatctctc tgatattagc gctcaattac cctctgactt 3060
tgttcagggt gttcagttaa ttctcccgtc taatgcgctt ccctgttttt atgttattct 3120
ctctgtaaag gctgctattt tcatttttga cgttaaacaa aaaatcgttt cttatttgga 3180
ttgggataaa taatatggct gtttattttg taactggcaa attaggctct ggaaagacgc 3240
tcgttagcgt tggtaagatt caggataaaa ttgtagctgg gtgcaaaata gcaactaatc 3300
ttgatttaag gcttcaaaac ctcccgcaag tcgggaggtt cgctaaaacg cctcgcgttc 3360
ttagaatacc ggataagcct tctatatctg atttgcttgc tattgggcgc ggtaatgatt 3420
cctacgatga aaataaaaac ggcttgcttg ttctcgatga gtgcggtact tggtttaata 3480
cccgttcttg gaatgataag gaaagacagc cgattattga ttggtttcta catgctcgta 3540
aattaggatg ggatattatt tttcttgttc aggacttatc tattgttgat aaacaggcgc 3600
gttctgcatt agctgaacat gttgtttatt gtcgtcgtct ggacagaatt actttacctt 3660
ttgtcggtac tttatattct cttattactg gctcgaaaat gcctctgcct aaattacatg 3720
ttggcgttgt taaatatggc gattctcaat taagccctac tgttgagcgt tggctttata 3780
ctggtaagaa tttgtataac gcatatgata ctaaacaggc tttttctagt aattatgatt 3840
ccggtgttta ttcttattta acgccttatt tatcacacgg tcggtatttc aaaccattaa 3900
atttaggtca gaagatgaaa ttaactaaaa tatatttgaa aaagttttct cgcgttcttt 3960
gtcttgcgat tggatttgca tcagcattta catatagtta tataacccaa cctaagccgg 4020
aggttaaaaa ggtagtctct cagacctatg attttgataa attcactatt gactcttctc 4080
agcgtcttaa tctaagctat cgctatgttt tcaaggattc taagggaaaa ttaattaata 4140
gcgacgattt acagaagcaa ggttattcac tcacatatat tgatttatgt actgtttcca 4200
ttaaaaaagg taattcaaat gaaattgtta aatgtaatta attttgtttt cttgatgttt 4260
gtttcatcat cttcttttgc tcaggtaatt gaaatgaata attcgcctct gcgcgatttt 4320
gtaacttggt attcaaagca atcaggcgaa tccgttattg tttctcccga tgtaaaaggt 4380
actgttactg tatattcatc tgacgttaaa cctgaaaatc tacgcaattt ctttatttct 4440
gttttacgtg caaataattt tgatatggta ggttctaacc cttccattat tcagaagtat 4500
aatccaaaca atcaggatta tattgatgaa ttgccatcat ctgataatca ggaatatgat 4560
gataattccg ctccttctgg tggtttcttt gttccgcaaa atgataatgt tactcaaact 4620
tttaaaatta ataacgttcg ggcaaaggat ttaatacgag ttgtcgaatt gtttgtaaag 4680
tctaatactt ctaaatcctc aaatgtatta tctattgacg gctctaatct attagttgtt 4740
agtgctccta aagatatttt agataacctt cctcaattcc tttcaactgt tgatttgcca 4800
actgaccaga tattgattga gggtttgata tttgaggttc agcaaggtga tgctttagat 4860
ttttcatttg ctgctggctc tcagcgtggc actgttgcag gcggtgttaa tactgaccgc 4920
ctcacctctg ttttatcttc tgctggtggt tcgttcggta tttttaatgg cgatgtttta 4980
gggctatcag ttcgcgcatt aaagactaat agccattcaa aaatattgtc tgtgccacgt 5040
attcttacgc tttcaggtca gaagggttct atctctgttg gccagaatgt cccttttatt 5100
actggtcgtg tgactggtga atctgccaat gtaaataatc catttcagac gattgagcgt 5160
caaaatgtag gtatttccat gagcgttttt cctgttgcaa tggctggcgg taatattgtt 5220
ctggatatta ccagcaaggc cgatagtttg agttcttcta ctcaggcaag tgatgttatt 5280
actaatcaaa gaagtattgc tacaacggtt aatttgcgtg atggacagac tcttttactc 5340
ggtggcctca ctgattataa aaacacttct caggattctg gcgtaccgtt cctgtctaaa 5400
atccctttaa tcggcctcct gtttagctcc cgctctgatt ctaacgagga aagcacgtta 5460
tacgtgctcg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 5520
tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 5580
cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 5640
ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 5700
tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 5760
gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 5820
tatctcgggc tattcttttg atttataagg gattttgccg atttcggaac caccatcaaa 5880
caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc 5940
caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg 6000
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 6060
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 6120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 6180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcgagct 6240
cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca ctggccgtcg 6300
ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 6360
atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 6420
agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc 6480
cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact 6540
ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca 6600
atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg 6660
atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt cctattggtt 6720
aaaaaatgag ctgatttaac aaaaatttaa tgcgaatttt aacaaaatat taacgtttac 6780
aatttaaata tttgcttata caatcttcct gtttttgggg cttttctgat tatcaaccgg 6840
ggtacatatg attgacatgc tagttttacg attaccgttc atcgattctc ttgtttgctc 6900
cagactctca ggcaatgacc tgatagcctt tgtagatctc tcaaaaatag ctaccctctc 6960
cggcattaat ttatcagcta gaacggttga atatcatatt gatggtgatt tgactgtctc 7020
cggcctttct cacccttttg aatctttacc tacacattac tcaggcattg catttaaaat 7080
atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc ccgcaaaagt 7140
attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg aggctttatt 7200
gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgtt 7249
<210> 174
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
tattattatt attattttt 19
<210> 175
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
tttttttttt tttttagcg 19

Claims (10)

1. The preparation method of the tweezer-shaped composite nano probe is characterized by comprising the following steps of:
A. synthesizing a gold nanorod, and modifying the surface of the gold nanorod with a specific sequence DNA with a mercapto functional group;
B. synthesizing a DNA origami with a specific capture chain at a pre-designed site;
C. assembling the gold nanorods modified in the step A and DNA origami to obtain a tweezers-shaped composite nanoprobe;
the specific sequence DNA with the sulfhydryl functional group in the step A is as follows: 5 '-TATTATTATTATTATTTTT-SH-3' and 5 '-TTTTTTTTTTTTTTTAGCG-SH-3';
the DNA origami in the step B is assembled by 1M 13mp18 bacteriophage long-chain DNA and 172 short-chain DNA through base complementary pairing, wherein the 172 short-chain DNA comprises 32 capture chains and auxiliary folding chains, and the nucleic acid sequence of the capture chains is shown as SEQ ID NO. 1-32; the nucleic acid sequence of the auxiliary folding strand is shown as SEQ ID NO. 33-172; the nucleic acid sequence of the long-chain DNA is shown as SEQ ID NO. 173;
and C, hybridizing the modified gold nanorods with the capture chains on the DNA origami through the specific sequence DNA with the mercapto functional groups modified on the surfaces of the gold nanorods, and assembling to obtain the tweezers-shaped composite nanoprobe.
2. The method of claim 1, wherein step a comprises:
a1, synthesizing seed crystal solution;
a2, growing gold nanorods by using the seed crystals synthesized in the step A1 as a substrate;
a3, purifying the gold nanorods obtained in the step A2;
a4, modifying the DNA with a special sequence of a sulfhydryl functional group on the surface of the gold nanorod.
3. The method of claim 2, wherein step a1 comprises: adding 50 mu l of 2% chloroauric acid solution into 9.5ml CTAB solution, stirring uniformly, adding 1ml of 6mM sodium borohydride solution under the condition of stirring, and carrying out water bath at 30 ℃ for 6-12h to obtain a seed crystal solution.
4. The method of claim 2, wherein step a2 comprises: adding 100ml of CTAB solution into a reactor, then adding 780 mu l of 2% chloroauric acid solution under stirring, adding 700 mu l of 10mM silver nitrate solution and 480 mu l of 100mM ascorbic acid solution under stirring, then adding 160 mu l of seed crystal solution prepared in the step A1, uniformly stirring, and carrying out water bath at 30 ℃ for 12 hours to obtain a colloidal solution containing gold nanorods;
step a3 includes: and D, centrifuging the colloidal solution obtained in the step A2 at 3000rpm for 20min, and removing the precipitate to obtain the purified gold nanorods.
5. The method of claim 2, wherein step a4 comprises:
(1) adding water into artificially synthesized DNA with a specific sequence of a sulfhydryl functional group to prepare DNA solutions of 100 mu M respectively, and adding a proper amount of TCEP until the final concentration is 20 mM;
(2) mixing 800 μ l of 5 XTBE buffer solution, 40 μ l of 1% SDS solution and 400 μ l of 5M NaCl solution, adding water to dilute to total volume of 4ml, and adjusting pH to 3.6 with hydrochloric acid to obtain modification solution;
(3) under the shaking condition, adding 100 mu l of the 100 mu M solution obtained in the step (1) into the modification solution, adding the gold nanorods purified in the step A3 under the shaking condition, and carrying out water bath at 30 ℃ for 6h to obtain modified gold nanorods; wherein, the mol ratio of the gold nanorods to each DNA is 1:2500 respectively.
6. The method of claim 1, wherein step B comprises:
b1, artificially synthesizing M13mp18 phage long-chain DNA and 172 short-chain DNA, mixing the long-chain DNA and the short-chain DNA according to the molar ratio of 1: 5-1: 10, and adding Mg into the mixture2+Self-assembly is carried out in the 1 XTAE buffer solution;
the self-assembly conditions were: the PCR amplificator was set up with the following program: cooling from 95 deg.C to 65 deg.C, and maintaining each gradient for 5min at every 5 deg.C; cooling from 65 deg.C to 25 deg.C, and maintaining each gradient for 10min at each temperature of 1 deg.C;
b2, after the self-assembly is finished, the self-assembly product and Mg are contained2+Mixing the 1 XTAE buffer solution according to the volume ratio of 1:4, adding into a centrifugal column with molecular weight cutoff of 100KDa, and centrifuging at 5000rpm for 3 min;
b3, adding Mg into the centrifugal column2+Repeatedly centrifuging and washing the 1 XTAE buffer solution for 3 times to obtain the DNA origami;
wherein said Mg is contained2+The composition of the 1 XTAE buffer solution is: 0.04M Tris, 0.02M acetic acid, 0.003M EDTA and 0.0125M magnesium acetate, pH 8.0.
7. The method of claim 1, wherein step C comprises:
c1, mixing the modified gold nanorods with DNA origami according to the mol ratio of 4: 1-10: 1, and adding Mg2+Self-assembly is carried out in the 1 XTAE buffer solution;
the self-assembly conditions were: the PCR amplificator was set up with the following program: cooling from 45 deg.C to 25 deg.C, and maintaining each gradient for 5min at every 1 deg.C; when the temperature is kept for 5min at 25 ℃, the temperature is raised to 45 ℃ for the next cycle, and the reaction is finished for 6 cycles;
c2, after the self-assembly is finished, carrying out 0.5% -2% agarose gel electrophoresis on the self-assembly product, wherein the electrophoresis conditions are as follows: 85V, 1 h;
c3, cutting gel and recovering an electrophoresis product, namely the tweezer-shaped composite nano probe.
8. The tweezer-like composite nanoprobe prepared according to the method of any one of claims 1 to 7.
9. The composite nanoprobe of claim 8, wherein the gold nanorods on the composite nanoprobe have a length of 40 ± 4nm and a diameter of 10 ± 1 nm; the absorption peak of the ultraviolet spectrum is in the wavelength region of 760 +/-20 nm; and/or
And detecting the wavelength range of the composite nano probe by circular dichroism spectrum within 400-900 nm.
10. Use of the composite nanoprobe of claim 8 or 9 for detecting reduced glutathione.
CN201910848227.4A 2019-09-09 2019-09-09 Tweezer-shaped composite nano probe and preparation method and application thereof Active CN110643604B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910848227.4A CN110643604B (en) 2019-09-09 2019-09-09 Tweezer-shaped composite nano probe and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910848227.4A CN110643604B (en) 2019-09-09 2019-09-09 Tweezer-shaped composite nano probe and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN110643604A CN110643604A (en) 2020-01-03
CN110643604B true CN110643604B (en) 2021-04-09

Family

ID=69010343

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910848227.4A Active CN110643604B (en) 2019-09-09 2019-09-09 Tweezer-shaped composite nano probe and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN110643604B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113433100B (en) * 2021-05-25 2022-09-27 上海市公共卫生临床中心 Plasma tryptophan and albumin joint detection method based on photochemical reaction of DNA synthesized silver nanoclusters and tryptophan

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106770049B (en) * 2016-12-22 2020-04-14 南京邮电大学 Method for constructing Dolmen structure based on DNA paper folding template and nano gold rod
US11352254B2 (en) * 2017-04-06 2022-06-07 Technische Universität München Molecular machine

Also Published As

Publication number Publication date
CN110643604A (en) 2020-01-03

Similar Documents

Publication Publication Date Title
US20080177053A1 (en) DNA nanocage by self-organization of DNA and process for producing the same, and DNA nanotube and molecule carrier using the same
Liu et al. Improving sensitivity of gold nanoparticle based fluorescence quenching and colorimetric aptasensor by using water resuspended gold nanoparticle
Zheng et al. Rational construction of a DNA nanomachine for HIV nucleic acid ultrasensitive sensing
CN109563539A (en) Use the Single Molecule Detection of DNA nanotechnology or quantitative
TWI519774B (en) Metal particle for surface enhanced raman scattering and molecular sensing,and a method for producing the same
JP2001512474A (en) In vitro peptide or protein expression library
CN110643604B (en) Tweezer-shaped composite nano probe and preparation method and application thereof
Davis et al. Biomolecular inorganic materials chemistry
CN108103151A (en) A kind of method and its application based on the protein-bonded detection of nucleic acids of sequence-specific nucleic acid and parting
CN110551725A (en) anticoagulation DNA nano composite structure and preparation method and application thereof
CN103540651A (en) Nanometer gold complex as well as preparation and application thereof
Slocik et al. Sequenced defined biomolecules for nanomaterial synthesis, functionalization, and assembly
CN102203246A (en) Controllable assembly and disassembly of nanoparticle systems via protein and DNA agents
Su et al. A universal CRISPR/Cas12a-mediated AuNPs aggregation-based surface-enhanced Raman scattering (CRISPR/Cas-SERS) platform for virus gene detection
Xie et al. Visualizing of AuNPs protection aptamer from DNase I enzyme digestion based on Nanopipette and its use for Microcystin-LR detection
CN103014145B (en) Controllable distribution method of gold nanoparticles on DNA origami chip
Hassanpour et al. pDNA conjugated with citrate capped silver nanoparticles towards ultrasensitive bio-assay of haemophilus influenza in human biofluids: A novel optical biosensor
CN105699699B (en) A kind of sample preparation methods of single antibody molecular atoms force microscope imaging
Zhan et al. DNA tetrahedron-based CRISPR bioassay for treble-self-amplified and multiplex HPV-DNA detection with elemental tagging
Ko et al. Selective release of DNA nanostructures from DNA hydrogel
CN109477096B (en) DNA paper folding unit step-by-step assembling method
CN113278607A (en) Preparation method of circular single-stranded DNA integrated by nucleic acid aptamer and application of circular single-stranded DNA integrated by nucleic acid aptamer in DNA paper folding
CN116515844A (en) Migration body aptamer and screening method and application thereof
CN112439370B (en) Preparation method of graphene oxide fluorescence-enhanced functional nucleic acid hydrogel
CN114099694B (en) Thrombin-responsive DNA nano machine and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant