CN107988316A - Preparation method of DNA nanometer image probes for subcellular fraction scale cell common location and products thereof and application - Google Patents

Preparation method of DNA nanometer image probes for subcellular fraction scale cell common location and products thereof and application Download PDF

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Publication number
CN107988316A
CN107988316A CN201711216405.9A CN201711216405A CN107988316A CN 107988316 A CN107988316 A CN 107988316A CN 201711216405 A CN201711216405 A CN 201711216405A CN 107988316 A CN107988316 A CN 107988316A
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dna
artificial sequence
chain
subcellular fraction
common location
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何丹农
王萍
陈益
刘婷
徐艳
金彩虹
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Shanghai National Engineering Research Center for Nanotechnology Co Ltd
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Shanghai National Engineering Research Center for Nanotechnology Co Ltd
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    • G01N33/56966—Animal cells

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Abstract

The present invention proposes a kind of preparation method of DNA nanometer image probes for subcellular fraction scale cell common location and products thereof and application, replacement chain DNA 1 with fluorescence and 207 staple chains are equally dissolved into MilliQ water, the ultimate density for making every chain is 200nM, by M13mp18 single stranded DNAs（100nM）With 208 mixed short chains (200nM) with molar concentration rate 1:5‑1：10 ratio is blended in 1 × TAE Mg2+In solution, wherein the ultimate density of M13mp18 single stranded DNAs is 5 nM, and short chain ultimate density is 50nM；Mixed solution is put into PCR instrument, 95 DEG C of the extent of reaction of setting continues 3 minutes, and then for slow cooling to 4 DEG C, rate of temperature fall is 0.2 DEG C/10s.Small size and preferable biocompatibility due to DNA origami structures, the present invention have larger application prospect in subcellular fraction scale imaging, lesion detection and drug action mechanism research field.

Description

Preparation method for the DNA nanometer image probes of subcellular fraction scale cell common location And products thereof and application
Technical field
The present invention propose a kind of DNA nanometer image probes for subcellular fraction scale cell common location preparation method and its Product and application.It is single-stranded more particularly to the DNA that will be marked with specific fluorescent molecule, by self assembly, modify and rolled in DNA triangles On paper structure, become a kind of DNA image probes, for realizing the effect of organelle common location.Due to DNA origami structures Small size and preferable biocompatibility, the present invention have in subcellular fraction scale imaging, lesion detection and drug action mechanism research field Larger application prospect.
Background technology
The nano-probe for subcellular fraction scale imaging and organelle common location existing at present mainly has following a few classes： Nano-probe based on magnetic material, this kind of nano-probe is mainly using the compound of iron or iron as matrix；Based on nanogold and its The detection probe of compound；And based on other inorganic nano materials, the nano-probe of such as carbon nanotubes and its compound.This Class probe forms usually inorganic matter or comprising metallics, has certain bio-toxicity, histocompatbility is poor, and metabolism is tired Difficult and mechanism is unknown.Therefore, preparing a kind of new safe nano-probe just becomes the task of top priority.Study DNA Material is used for specific recognition DNA sequence dna（A kind of nano-probe and its application process for specific recognition DNA sequence dna, it is public The number of opening：CN107245524A）, or for unimolecule Genotyping（A kind of unimolecule based on DNA paper foldings probe specificity mark Methods of genotyping and its application）.Rarely has the research that DNA paper foldings nanostructured is used for the imaging of subcellular fraction common location.
DNA triangular nanos image probe is used for the organelle common location of subcellular fraction scale, not only biological compatibility to be good, nothing Cytotoxicity, and fluorescence molecule and some small peptide micromoleculars can be modified easily in DNA origami structures, and this is just Structure for from DNA target to nanometer image probe provides theoretical foundation, the research available for organelle targeting common location.Meanwhile Due to the perfect match of base, DNA materials can be self-assembled into the 3-d modelling with certain structure size, by regulating and controlling nanometer The size of structure and the modification of targeted molecular, this nanometer of image probe can be more prone to reach target site and clearly into Picture, has higher research significance and application value.
The content of the invention
For deficiency of the prior art in fluorescence imaging field, it is an object of the invention to：One kind is provided and is used for subcellular fraction scale The preparation method of the DNA nanometer image probes of cell common location.
Another object of the present invention is：The product that a kind of above-mentioned preparation method obtains is provided.
A further object of the present invention is：A kind of application of the said goods is provided.
The purpose of the present invention is what is be achieved through the following technical solutions：
A kind of preparation method of DNA nanometer image probes for subcellular fraction scale cell common location, includes the following steps：Have 208 staple chain short chains, wherein one replaces chain DNA short chain and 207 staple chains of other sequences number etc. with fluorescence It is dissolved into MilliQ water to amount, the ultimate density for making every chain is 200Nm；By M13mp18 single stranded DNAs（100nM）With it is mixed 208 short chains (200nM) with molar concentration rate 1:5-1：10 ratio is blended in 1 × TAE-Mg2+In solution, wherein, The ultimate density of M13mp18 single stranded DNAs is 5 nM, and short chain ultimate density is 50nM；Mixed solution is put into PCR instrument, 95 DEG C of the extent of reaction of setting continues 3 minutes, and then for slow cooling to 4 DEG C, rate of temperature fall is 0.2 DEG C/10s.
The method of the present invention redesigns specific alkali by modifying the fluorescence molecule that can be excited by near-infrared in DNA chain Basic sequence, enables DNA chain to be self-assembled into DNA triangles under certain condition, cannot be only used for cell and normally swallows nanometer The subcellular fraction scale imaging research of grain, moreover it is possible to targeted molecular is further modified, such as tumour penetrating peptide, the targeting point of target organelles Son.To strengthen its penetrability and targeting, for targeting common location research.The present invention is using DNA materials as molecular probe Matrix, has extraordinary biocompatibility, before medicine delivery, subcellular fraction common location imaging field have wide application Scape.
The fluorescent dye that the replacement with fluorescence connects in DNA1 is Cy5, Cy3, FITC, FAM.
The fluorescent DNA chain that carries is any one in 208 staple chains.Such as the replacement chain DNA with fluorescence is During DNA 1,207 staple chains of itself and sequence number 2 to 208 are equally dissolved into MilliQ water.
The present invention provides one kind and is used for subcellular fraction scale DNA nanometers of image probes of cell common location, according to any of the above-described institute The method of stating is prepared.
Present invention also offers the application that a kind of DNA nanometers of image probe is used for subcellular fraction scale cell common location, by DNA Nanometer image probe is added in cell culture fluid is incubated culture with cell altogether, different thin according to the arrival of incubation time different probe Born of the same parents' device, after culture, discards the nutrient solution containing DNA nanometers of image probes, and residual culture, Ran Houyong are rinsed out with PBS Paraformaldehyde solution fixes cell, is rinsed again with PBS solution, adds 0.1% TritonX-100,10 minutes, thin to increase After birth permeability, is rinsed with PBS solution again, is added 2%BSA solution closing nonspecific activity site, is then added organelle Specific primary antibody is incubated, and discards primary antibody, and PBS is rinsed, and adds the secondary antibody with fluorescence, and room temperature lucifuge is incubated, and PBS is rinsed again, Finally plus anti-fluorescent quenching mounting liquid, it is placed in laser confocal microscope observation.
The organelle-specificity primary antibody is early endosome primary antibody, late endosome primary antibody, lysosome primary antibody, Gorky Body primary antibody, nucleus primary antibody.
Small size and preferable biocompatibility due to DNA origami structures, the present invention is in subcellular fraction scale imaging, tumour Detection and drug action mechanism research field have larger application prospect.
Compared with prior art, the invention has the advantages that：
1st, the present invention uses DNA materials, different fluorescence is connected by design dna sequence interchangeable, and be easily attached other Targeted molecular, it is possible to achieve the fluoroscopic examination of multi-wavelength, and the imaging of different targeting cell and crganelles；
2nd, the present invention can be more flexibly controllable by varying the structure regulating probe degradation time in vivo of DNA, use scope；
3rd, the DNA materials that the present invention uses, have extraordinary biocompatibility, avoid because nano-probe aggregation may make Into in vivo functionality area infringement.
Brief description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, further feature of the invention, Objects and advantages will become more apparent upon：
Attached drawing 1 is DNA nanostructure electrophoretogram, and L1 is staple chain DNA, and L2 is DNA triangle origami structures；
Attached drawing 2 for DNA nanometers of image probe cell in vitro be incubated altogether 12 it is small when laser confocal microscope image.
Embodiment
With reference to specific embodiment, the present invention is described in detail.Following embodiments will be helpful to the technology of this area Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill to this area For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention Protection domain.
Embodiment 1
207 staple chains of replacement chain DNA 1 with fluorescence Cy5 and DNA2-208 are equally dissolved into MilliQ water In, the ultimate density for making every chain is 200nM；By M13mp18 single stranded DNAs（100nM）With 208 mixed short chains (200nM) With molar concentration rate 1:5 ratio is blended in 1 × TAE-Mg2+In solution, wherein the ultimate density of M13mp18 single stranded DNAs is 5 NM, short chain ultimate density are 50nM.Mixed solution is put into PCR instrument, 95 DEG C of the extent of reaction of setting continues 3 minutes, then For slow cooling to 4 DEG C, rate of temperature fall is 0.2 DEG C/10s.Obtain DNA nanometers of image probes.
DNA nanometers image probe prepared in above-mentioned steps is added in cell culture fluid and is incubated culture 12 altogether with cell Hour.After culture, the nutrient solution containing DNA nanometers of image probes is discarded, residual culture is rinsed out with PBS.Then use Paraformaldehyde solution fixes cell, is rinsed again with PBS solution, adds 0.1% TritonX-100,10 minutes, thin to increase After birth permeability.Rinsed 3 times with PBS solution again, add 2%BSA solution closing nonspecific activity site.Then add early Phase endosome primary antibody solution is incubated, and discards primary antibody, and PBS is rinsed, and adds the secondary antibody with green fluorescence, and room temperature lucifuge is incubated.Again Secondary PBS is rinsed, and finally adds anti-fluorescent quenching mounting liquid.It is placed in laser confocal microscope observation.
Embodiment 2
207 staple chains of the replacement chain DNA 1 with fluorescence Cy3 and other sequence numbers are equally dissolved into MilliQ water In, the ultimate density for making every chain is 200nM.By M13mp18 single stranded DNAs（100nM）With 208 mixed short chains (200nM) With molar concentration rate 1:10 ratio is blended in 1 × TAE-Mg2+In solution, wherein the ultimate density of M13mp18 single stranded DNAs is 5 NM, short chain ultimate density are 50nM.Mixed solution is put into PCR instrument, 95 DEG C of the extent of reaction of setting continues 3 minutes, then For slow cooling to 4 DEG C, rate of temperature fall is 0.2 DEG C/10s, obtains DNA nanometers of image probes.
DNA nanometers image probe prepared in above-mentioned steps is added in cell culture fluid and is incubated culture 10 altogether with cell Hour.After culture, the nutrient solution containing DNA nanometers of image probes is discarded, residual culture is rinsed out with PBS.Then use Paraformaldehyde solution fixes cell, is rinsed again with PBS solution, adds 0.1% TritonX-100,10 minutes, thin to increase After birth permeability.Rinsed 3 times with PBS solution again, add 2%BSA solution closing nonspecific activity site.Then evening is added Phase endosome primary antibody solution is incubated, and discards primary antibody, and PBS is rinsed, and adds the secondary antibody with green fluorescence, and room temperature lucifuge is incubated.Again Secondary PBS is rinsed, and finally adds anti-fluorescent quenching mounting liquid.It is placed in laser confocal microscope observation.
Embodiment 3
207 staple chains of the replacement chain DNA 1 with fluorescence FITC and other sequences number are equally dissolved into MilliQ In water, the ultimate density for making every chain is 200nM.By M13mp18 single stranded DNAs（100nM）With 208 mixed short chains (200nM) is with molar concentration rate 1:7 ratio is blended in 1 × TAE-Mg2+In solution, wherein M13mp18 single stranded DNAs is final Concentration is 5 nM, and short chain ultimate density is 50nM.Mixed solution is put into PCR instrument, 95 DEG C of the extent of reaction of setting continues 3 Minute, then for slow cooling to 4 DEG C, rate of temperature fall is 0.2 DEG C/10s, obtains DNA nanometers of image probes.
DNA nanometers image probe prepared in above-mentioned steps is added in cell culture fluid and is incubated culture 2 altogether with cell Hour.After culture, the nutrient solution containing DNA nanometers of image probes is discarded, residual culture is rinsed out with PBS.Then use Paraformaldehyde solution fixes cell, is rinsed again with PBS solution, adds 0.1% TritonX-100,10 minutes, thin to increase After birth permeability.Rinsed 3 times with PBS solution again, add 2%BSA solution closing nonspecific activity site.Then add molten Enzyme body primary antibody solution is incubated, and discards primary antibody, and PBS is rinsed, and adds the secondary antibody with red fluorescence, and room temperature lucifuge is incubated.PBS again Rinse, finally add anti-fluorescent quenching mounting liquid.It is placed in laser confocal microscope observation.
Embodiment 4
207 staple chains of the replacement chain DNA 1 with fluorescence FAM and other sequences number are equally dissolved into MilliQ water In, the ultimate density for making every chain is 200nM.By M13mp18 single stranded DNAs（100nM）With 208 mixed short chains (200nM) With molar concentration rate 1:9 ratio is blended in 1 × TAE-Mg2+In solution, wherein the ultimate density of M13mp18 single stranded DNAs is 5 NM, short chain ultimate density are 50nM.Mixed solution is put into PCR instrument, 95 DEG C of the extent of reaction of setting continues 3 minutes, then For slow cooling to 4 DEG C, rate of temperature fall is 0.2 DEG C/10s, obtains DNA nanometers of image probes.
DNA nanometers image probe prepared in above-mentioned steps is added in cell culture fluid and is incubated culture 24 altogether with cell Hour.After culture, the nutrient solution containing DNA nanometers of image probes is discarded, residual culture is rinsed out with PBS.Then use Paraformaldehyde solution fixes cell, is rinsed again with PBS solution, adds 0.1% TritonX-100,10 minutes, thin to increase After birth permeability.Rinsed 3 times with PBS solution again, add 2%BSA solution closing nonspecific activity site.Then add high Dictyosome primary antibody solution is incubated, and discards primary antibody, and PBS is rinsed, and adds the secondary antibody with red fluorescence, and room temperature lucifuge is incubated.Again PBS is rinsed, and finally adds anti-fluorescent quenching mounting liquid.It is placed in laser confocal microscope observation.
The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow Ring the substantive content of the present invention.
<110>Shanghai National Engineering Research Center for Nanotechnology Co., Ltd
<120>Preparation method of DNA nanometer image probes for subcellular fraction scale cell common location and products thereof and application
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actagaaata tataactata tgtacgctga ga
<210>44
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<210>45
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<210>55
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<210>62
<211>24
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tcagctaaaa aaggtaaagt aatt
<210>63
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acgctaacga gcgtctggcg ttttagcgaa cccaacatgt
<210>64
<211>43
<212>DNA
<213>Artificial sequence
<400>
acgacaataa atcccgactt gcgggagatc ctgaatctta cca
<210>65
<211>43
<212>DNA
<213>Artificial sequence
<400>
tgctattttg cacccagcta caattttgtt ttgaagcctt aaa
<210>66
<211>32
<212>DNA
<213>Artificial sequence
<400>
tcatatgtgt aatcgtaaaa ctagtcattt tc
<210>67
<211>32
<212>DNA
<213>Artificial sequence
<400>
gtgagaaaat gtgtaggtaa agatacaact tt
<210>68
<211>32
<212>DNA
<213>Artificial sequence
<400>
ggcatcaaat ttggggcgcg agctagttaa ag
<210>69
<211>32
<212>DNA
<213>Artificial sequence
<400>
ttcgagctaa gacttcaaat atcgggaacg ag
<210>70
<211>40
<212>DNA
<213>Artificial sequence
<400>
acagtcaaag agaatcgatg aacgaccccg gttgataatc
<210>71
<211>32
<212>DNA
<213>Artificial sequence
<400>
atagtagtat gcaatgcctg agtaggccgg ag
<210>72
<211>32
<212>DNA
<213>Artificial sequence
<400>
aaccagacgt ttagctatat tttcttctac ta
<210>73
<211>40
<212>DNA
<213>Artificial sequence
<400>
gaataccaca ttcaacttaa gaggaagccc gatcaaagcg
<210>74
<211>40
<212>DNA
<213>Artificial sequence
<400>
agaaaagccc caaaaagagt ctggagcaaa caatcaccat
<210>75
<211>32
<212>DNA
<213>Artificial sequence
<400>
caatatgacc ctcatatatt ttaaagcatt aa
<210>76
<211>32
<212>DNA
<213>Artificial sequence
<400>
catccaataa atggtcaata acctcggaag ca
<210>77
<211>40
<212>DNA
<213>Artificial sequence
<400>
aactccaaga ttgcatcaaa aagataatgc agatacataa
<210>78
<211>40
<212>DNA
<213>Artificial sequence
<400>
cgttctagtc aggtcattgc ctgacaggaa gattgtataa
<210>79
<211>32
<212>DNA
<213>Artificial sequence
<400>
caggcaagat aaaaattttt agaatattca ac
<210>80
<211>32
<212>DNA
<213>Artificial sequence
<400>
gattagagat tagatacatt tcgcaaatca ta
<210>81
<211>40
<212>DNA
<213>Artificial sequence
<400>
cgccaaaagg aattacagtc agaagcaaag cgcaggtcag
<210>82
<211>40
<212>DNA
<213>Artificial sequence
<400>
gcaaatattt aaattgagat ctacaaaggc tactgataaa
<210>83
<211>32
<212>DNA
<213>Artificial sequence
<400>
ttaatgcctt atttcaacgc aagggcaaag aa
<210>84
<211>32
<212>DNA
<213>Artificial sequence
<400>
ttagcaaata gatttagttt] gaccagtacc tt
<210>85
<211>40
<212>DNA
<213>Artificial sequence
<400>
taattgcttt accctgacta ttatgaggca tagtaagagc
<210>86
<211>35
<212>DNA
<213>Artificial sequence
<400>
ataaagcctt tgcgggagaa gcctggagag ggtag
<210>87
<211>32
<212>DNA
<213>Artificial sequence
<400>
taagaggtca attctgcgaa cgagattaag ca
<210>88
<211>40
<212>DNA
<213>Artificial sequence
<400>
aacactatca taacccatca aaaatcaggt ctccttttga
<210>89
<211>24
<212>DNA
<213>Artificial sequence
<400>
atgaccctgt aatacttcag agca
<210>90
<211>32
<212>DNA
<213>Artificial sequence
<400>
taaagctata taacagttga ttcccatttt tg
<210>91
<211>40
<212>DNA
<213>Artificial sequence
<400>
cggatggcac gagaatgacc ataatcgttt accagacgac
<210>92
<211>35
<212>DNA
<213>Artificial sequence
<400>
taattgcttg gaagtttcat tccaaatcgg ttgta
<210>93
<211>40
<212>DNA
<213>Artificial sequence
<400>
gataaaaacc aaaatattaa acagttcaga aattagagct
<210>94
<211>24
<212>DNA
<213>Artificial sequence
<400>
actaaagtac ggtgtcgaat ataa
<210>95
<211>40
<212>DNA
<213>Artificial sequence
<400>
tgctgtagat ccccctcaaa tgctgcgaga ggcttttgca
<210>96
<211>43
<212>DNA
<213>Artificial sequence
<400>
aaagaagttt tgccagcata aatattcatt gactcaacat gtt
<210>97
<211>43
<212>DNA
<213>Artificial sequence
<400>
aatactgcgg aatcgtaggg ggtaatagta aaatgtttag act
<210>98
<211>32
<212>DNA
<213>Artificial sequence
<400>
agggatagct cagagccacc accccatgtc aa
<210>99
<211>32
<212>DNA
<213>Artificial sequence
<400>
caacagttta tgggattttg ctaatcaaaa gg
<210>100
<211>32
<212>DNA
<213>Artificial sequence
<400>
gccgctttgc tgaggcttgc aggggaaaag gt
<210>101
<211>32
<212>DNA
<213>Artificial sequence
<400>
gcgcagactc catgttactt agcccgtttt aa
<210>102
<211>32
<212>DNA
<213>Artificial sequence
<400>
acaggtagaa agattcatca gttgagattt ag
<210>103
<211>40
<212>DNA
<213>Artificial sequence
<400>
cctcagaacc gccacccaag cccaatagga acgtaaatga
<210>104
<211>32
<212>DNA
<213>Artificial sequence
<400>
attttctgtc agcggagtga gaataccgat at
<210>105
<211>32
<212>DNA
<213>Artificial sequence
<400>
attcggtctg cgggatcgtc acccgaaatc cg
<210>106
<211>40
<212>DNA
<213>Artificial sequence
<400>
cgacctgcgg tcaatcataa gggaacggaa caacattatt
<210>107
<211>40
<212>DNA
<213>Artificial sequence
<400>
agacgttacc atgtaccgta acacccctca gaaccgccac
<210>108
<211>32
<212>DNA
<213>Artificial sequence
<400>
cacgcataag aaaggaacaa ctaagtcttt cc
<210>109
<211>32
<212>DNA
<213>Artificial sequence
<400>
attgtgtctc agcagcgaaa gacaccatcg cc
<210>110
<211>40
<212>DNA
<213>Artificial sequence
<400>
ttaataaaac gaactaaccg aactgaccaa ctcctgataa
<210>111
<211>40
<212>DNA
<213>Artificial sequence
<400>
aggtttagta ccgccatgag tttcgtcacc aggatctaaa
<210>112
<211>32
<212>DNA
<213>Artificial sequence
<400>
gttttgtcag gaattgcgaa taatccgaca at
<210>113
<211>32
<212>DNA
<213>Artificial sequence
<400>
gacaacaagc atcggaacga gggtgagatt tg
<210>114
<211>40
<212>DNA
<213>Artificial sequence
<400>
tatcatcgtt gaaagaggac agatggaaga aaaatctacg
<210>115
<211>40
<212>DNA
<213>Artificial sequence
<400>
agcgtaacta caaactacaa cgcctatcac cgtactcagg
<210>116
<211>32
<212>DNA
<213>Artificial sequence
<400>
tagttgcgaa ttttttcacg ttgatcatag tt
<210>117
<211>32
<212>DNA
<213>Artificial sequence
<400>
gtacaacgag caacggctac agaggatacc ga
<210>118
<211>40
<212>DNA
<213>Artificial sequence
<400>
accagtcagg acgttggaac ggtgtacaga ccgaaacaaa
<210>119
<211>35
<212>DNA
<213>Artificial sequence
<400>
acagacagcc caaatctcca aaaaaaaatt tctta
<210>120
<211>32
<212>DNA
<213>Artificial sequence
<400>
aacagcttgc tttgaggact aaagcgatta ta
<210>121
<211>40
<212>DNA
<213>Artificial sequence
<400>
ccaagcgcag gcgcataggc tggcagaact ggctcattat
<210>122
<211>24
<212>DNA
<213>Artificial sequence
<400>
cgaggtgagg ctccaaaagg agcc
<210>123
<211>32
<212>DNA
<213>Artificial sequence
<400>
acccccagac tttttcatga ggaacttgct tt
<210>124
<211>40
<212>DNA
<213>Artificial sequence
<400>
accttatgcg attttatgac cttcatcaag agcatctttg
<210>125
<211>35
<212>DNA
<213>Artificial sequence
<400>
cggtttatca ggtttccatt aaacgggaat acact
<210>126
<211>40
<212>DNA
<213>Artificial sequence
<400>
aaaacactta atcttgacaa gaacttaatc attgtgaatt
<210>127
<211>24
<212>DNA
<213>Artificial sequence
<400>
ggcaaaagta aaatacgtaa tgcc
<210>128
<211>40
<212>DNA
<213>Artificial sequence
<400>
tggtttaatt tcaactcgga tattcattac ccacgaaaga
<210>129
<211>43
<212>DNA
<213>Artificial sequence
<400>
accaacctaa aaaatcaacg taacaaataa attgggcttg aga
<210>130
<211>43
<212>DNA
<213>Artificial sequence
<400>
cctgacgaga aacaccagaa cgagtaggct gctcattcag tga
<210>131
<211>32
<212>DNA
<213>Artificial sequence
<400>
tcgggagata tacagtaaca gtacaaataa tt
<210>132
<211>32
<212>DNA
<213>Artificial sequence
<400>
cctgattaaa ggagcggaat tatctcggcc tc
<210>133
<211>32
<212>DNA
<213>Artificial sequence
<400>
gcaaatcacc tcaatcaata tctgcaggtc ga
<210>134
<211>32
<212>DNA
<213>Artificial sequence
<400>
cgaccagtac attggcagat tcacctgatt gc
<210>135
<211>40
<212>DNA
<213>Artificial sequence
<400>
tggcaatttt taacgtcaga tgaaaacaat aacggattcg
<210>136
<211>32
<212>DNA
<213>Artificial sequence
<400>
aaggaattac aaagaaacca ccagtcagat ga
<210>137
<211>32
<212>DNA
<213>Artificial sequence
<400>
ggacattcac ctcaaatatc aaacacagtt ga
<210>138
<211>40
<212>DNA
<213>Artificial sequence
<400>
ttgacgagca cgtatactga aatggattat ttaataaaag
<210>139
<211>40
<212>DNA
<213>Artificial sequence
<400>
cctgattgct ttgaattgcg tagattttca ggcatcaata
<210>140
<211>32
<212>DNA
<213>Artificial sequence
<400>
taatcctgat tatcattttg cggagaggaa gg
<210>141
<211>32
<212>DNA
<213>Artificial sequence
<400>
ttatctaaag catcaccttg ctgatggcca ac
<210>142
<211>40
<212>DNA
<213>Artificial sequence
<400>
agagatagtt tgacgctcaa tcgtacgtgc tttcctcgtt
<210>143
<211>40
<212>DNA
<213>Artificial sequence
<400>
gattatacac agaaataaag aaataccaag ttacaaaatc
<210>144
<211>32
<212>DNA
<213>Artificial sequence
<400>
taggagcata aaagtttgag taacattgtt tg
<210>145
<211>32
<212>DNA
<213>Artificial sequence
<400>
tgacctgaca aatgaaaaat ctaaaatatc tt
<210>146
<211>40
<212>DNA
<213>Artificial sequence
<400>
agaatcagag cgggagatgg aaatacctac ataacccttc
<210>147
<211>40
<212>DNA
<213>Artificial sequence
<400>
gcgcagaggc gaattaatta tttgcacgta aattctgaat
<210>148
<211>32
<212>DNA
<213>Artificial sequence
<400>
aatggaagcg aacgttatta atttctaaca ac
<210>149
<211>32
<212>DNA
<213>Artificial sequence
<400>
taatagatcg ctgagagcca gcagaagcgt aa
<210>150
<211>40
<212>DNA
<213>Artificial sequence
<400>
gaatacgtaa caggaaaaac gctcctaaac aggaggccga
<210>151
<211>35
<212>DNA
<213>Artificial sequence
<400>
tcaatagata ttaaatcctt tgccggttag aacct
<210>152
<211>32
<212>DNA
<213>Artificial sequence
<400>
caatatttgc ctgcaacagt gccatagagc cg
<210>153
<211>40
<212>DNA
<213>Artificial sequence
<400>
ttaaagggat tttagatacc gccagccatt gcggcacaga
<210>154
<211>24
<212>DNA
<213>Artificial sequence
<400>
acaattcgac aactcgtaat acat
<210>155
<211>32
<212>DNA
<213>Artificial sequence
<400>
ttgaggatgg tcagtattaa caccttgaat gg
<210>156
<211>40
<212>DNA
<213>Artificial sequence
<400>
ctattagtat atccagaaca atatcaggaa cggtacgcca
<210>157
<211>35
<212>DNA
<213>Artificial sequence
<400>
cgcgaactaa aacagaggtg aggcttagaa gtatt
<210>158
<211>40
<212>DNA
<213>Artificial sequence
<400>
gaatcctgag aagtgtatcg gccttgctgg tactttaatgTAATG
<210>159
<211>24
<212>DNA
<213>Artificial sequence
<400>
accaccagca gaagatgata gccc
<210>160
<211>40
<212>DNA
<213>Artificial sequence
<400>
taaaacatta gaagaactca aactttttat aatcagtgag
<210>161
<211>43
<212>DNA
<213>Artificial sequence
<400>
gccaccgagt aaaagaacat cacttgcctg agcgccatta aaa
<210>162
<211>43
<212>DNA
<213>Artificial sequence
<400>
tctttgatta gtaatagtct gtccatcacg caaattaacc gtt
<210>163
<211>32
<212>DNA
<213>Artificial sequence
<400>
cgcgtcrgat aggaacgcca tcaactttta ca
<210>164
<211>32
<212>DNA
<213>Artificial sequence
<400>
aggaagatgg ggacgacgac agtaatcata tt
<210>165
<211>32
<212>DNA
<213>Artificial sequence
<400>
ctctagagca agcttgcatg cctggtcagt tg
<210>166
<211>32
<212>DNA
<213>Artificial sequence
<400>
ccttcaccgt gagacgggca acagcagtca ca
<210>167
<211>32
<212>DNA
<213>Artificial sequence
<400>
cgagaaagga agggaagcgt actatggttg ct
<210>168
<211>40
<212>DNA
<213>Artificial sequence
<400>
gctcattttt taaccagcct tcctgtagcc aggcatctgc
<210>169
<211>32
<212>DNA
<213>Artificial sequence
<400>
cagtttgacg cactccagcc agctaaacga cg
<210>170
<211>32
<212>DNA
<213>Artificial sequence
<400>
gccattgcga tccccgggta ccgagttttt ct
<210>171
<211>40
<212>DNA
<213>Artificial sequence
<400>
tttcaccagc ctggccctga gagaaagccg gcgaacgtgg
<210>172
<211>40
<212>DNA
<213>Artificial sequence
<400>
gtaaccgtct ttcatcaaca ttaaaatttt tgttaaatca
<210>173
<211>32
<212>DNA
<213>Artificial sequence
<400>
acgttgtatt ccggcaccgc ttctggcgca tc
<210>174
<211>32
<212>DNA
<213>Artificial sequence
<400>
ccagggtggc tcgaattcgt aatccagtca cg
<210>175
<211>40
<212>DNA
<213>Artificial sequence
<400>
tagagcttga cggggagttg cagcaagcgg tcattgggcg
<210>176
<211>40
<212>DNA
<213>Artificial sequence
<400>
gttaaaattc gcattaatgt gagcgagtaa cacacgttgg
<210>177
<211>32
<212>DNA
<213>Artificial sequence
<400>
tgtagatggg tgccggaaac caggaacgcc ag
<210>178
<211>32
<212>DNA
<213>Artificial sequence
<400>
ggttttccat ggtcatagct gtttgagagg cg
<210>179
<211>40
<212>DNA
<213>Artificial sequence
<400>
gtttgcgtca cgctggtttg ccccaaggga gcccccgatt
<210>180
<211>40
<212>DNA
<213>Artificial sequence
<400>
ggataggtac ccgtcggatt ctcctaaacg ttaatatttt
<210>181
<211>32
<212>DNA
<213>Artificial sequence
<400>
agttgggtca aagcgccatt cgccccgtaa tg
<210>182
<211>32
<212>DNA
<213>Artificial sequence
<400>
cgcgcgggcc tgtgtgaaat tgttggcgat ta
<210>183
<211>40
<212>DNA
<213>Artificial sequence
<400>
ctaaatcgga accctaagca ggcgaaaatc cttcggccaa
<210>184
<211>35
<212>DNA
<213>Artificial sequence
<400>
cggcggattg aattcaggct gcgcaacggg ggatg
<210>185
<211>32
<212>DNA
<213>Artificial sequence
<400>
tgctgcaaat ccgctcacaa ttcccagctg ca
<210>186
<211>40
<212>DNA
<213>Artificial sequence
<400>
ttaatgaagt ttgatggtgg ttccgaggtg ccgtaaagca
<210>187
<211>24
<212>DNA
<213>Artificial sequence
<400>
tggcgaaatg ttgggaaggg cgat
<210>188
<211>32
<212>DNA
<213>Artificial sequence
<400>
tatcgtgcac acaacatacg agccacgcca gc
<210>189
<211>40
<212>DNA
<213>Artificial sequence
<400>
caagtttttt ggggtcgaaa tcggcaaaat ccgggaaacc
<210>190
<211>35
<212>DNA
<213>Artificial sequence
<400>
tcttcgctat tggaagcata aagtgtatgc ccgct
<210>191
<211>40
<212>DNA
<213>Artificial sequence
<400>
ttccagtcct tataaatcaa aagagaacca tcacccaaat
<210>192
<211>24
<212>DNA
<213>Artificial sequence
<400>
gcgctcacaa gcctggggtg ccta
<210>193
<211>40
<212>DNA
<213>Artificial sequence
<400>
cgatggccca catcgtatag cccgagatag ggattgcgtt
<210>194
<211>43
<212>DNA
<213>Artificial sequence
<400>
aactcacatt attgagtgtt gttccagaaa ccgtctatca ggg
<210>195
<211>43
<212>DNA
<213>Artificial sequence
<400>
acgtggactc caacgtcaaa gggcgaattt ggaacaagag tcc
<210>196
<211>25
<212>DNA
<213>Artificial sequence
<400>
ttaattaatt ttttaccata tcaaa
<210>197
<211>24
<212>DNA
<213>Artificial sequence
<400>
ttaatttcat cttagacttt acaa
<210>198
<211>23
<212>DNA
<213>Artificial sequence
<400>
ctgtccagac gtataccgaa cga
<210>199
<211>22
<212>DNA
<213>Artificial sequence
<400>
tcaagattag tgtagcaata ct
<210>200
<211>25
<212>DNA
<213>Artificial sequence
<400>
tgtagcattc cttttataaa cagtt
<210>201
<211>24
<212>DNA
<213>Artificial sequence
<400>
tttaattgta tttccaccag agcc
<210>202
<211>23
<212>DNA
<213>Artificial sequence
<400>
actacgaagg cttagcacca tta
<210>203
<211>22
<212>DNA
<213>Artificial sequence
<400>
ataaggcttg caacaaagtt ac
<210>204
<211>25
<212>DNA
<213>Artificial sequence
<400>
gtgggaacaa atttctattt ttgag
<210>205
<211>24
<212>DNA
<213>Artificial sequence
<400>
cggtgcgggc cttccaaaaa catt
<210>206
<211>23
<212>DNA
<213>Artificial sequence
<400>
atgagtgagc ttttaaatat gca
<210>207
<211>22
<212>DNA
<213>Artificial sequence
<400>
actattaaag aggatagcgt cc
<210>208
<211>24
<212>DNA
<213>Artificial sequence
<400>
gcgcttaatg cgccgctaca gggc

Claims (6)

  1. A kind of 1. preparation method of DNA nanometer image probes for subcellular fraction scale cell common location, it is characterised in that including Following steps：There are 208 staple chain short chains, wherein 207 of a replacement chain DNA short chain and other sequences number with fluorescence Staple chain is equally dissolved into MilliQ water, and the ultimate density for making every chain is 200Nm；By M13mp18 single stranded DNAs （100nM）With 208 mixed short chains (200nM) with molar concentration rate 1:5-1：10 ratio is blended in 1 × TAE-Mg2+Solution In, wherein, the ultimate density of M13mp18 single stranded DNAs is 5 nM, and short chain ultimate density is 50nM；Mixed solution is put into In PCR instrument, 95 DEG C of the extent of reaction of setting continues 3 minutes, and then for slow cooling to 4 DEG C, rate of temperature fall is 0.2 DEG C/10s.
  2. 2. it is used for the preparation method of the DNA nanometer image probes of subcellular fraction scale cell common location according to claim 1, its It is characterized in that, the fluorescent dye that the replacement with fluorescence connects in DNA1 is Cy5, Cy3, FITC, FAM.
  3. 3. it is used for the preparation method of the DNA nanometer image probes of subcellular fraction scale cell common location according to claim 1, its It is characterized in that, when the replacement chain DNA short chain with fluorescence is DNA1,207 staplers with other DNA sequence dnas 2-208 Nail chain is equally dissolved into MilliQ water.
  4. 4. one kind is used for subcellular fraction scale DNA nanometers of image probes of cell common location, it is characterised in that is appointed according to claim 1-3 One the method is prepared.
  5. 5. DNA nanometers of image probes are used for the application of subcellular fraction scale cell common location according to claim 4, its feature exists In DNA nanometers of image probes are added in cell culture fluids and be incubated culture altogether with cell, according to the arrival of incubation time different probe Different organelles, after culture, discards the nutrient solution containing DNA nanometers of image probes, and remaining culture is rinsed out with PBS Liquid, then fixes cell with paraformaldehyde solution, is rinsed again with PBS solution, adds 0.1% TritonX-100,10 minutes, To increase permeability of cell membrane, rinsed again with PBS solution, add 2%BSA solution closing nonspecific activity site, Ran Houjia Enter the incubation of organelle-specificity primary antibody, discard primary antibody, PBS is rinsed, and adds the secondary antibody with fluorescence, and room temperature lucifuge is incubated, again PBS is rinsed, and finally adds anti-fluorescent quenching mounting liquid, is placed in laser confocal microscope observation.
  6. 6. DNA nanometers of image probes are used for the application of subcellular fraction scale cell common location according to claim 5, its feature exists In, the organelle-specificity primary antibody be early endosome primary antibody, late endosome primary antibody, lysosome primary antibody, golgiosome one Anti- or nucleus primary antibody.
CN201711216405.9A 2017-11-28 2017-11-28 Preparation method of DNA nanometer image probes for subcellular fraction scale cell common location and products thereof and application Pending CN107988316A (en)

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