CN108624620A - DNA nanometers of tetrad realizations are efficiently conveyed to nucleic acid and the super sensitivity detection method of miRNA - Google Patents
DNA nanometers of tetrad realizations are efficiently conveyed to nucleic acid and the super sensitivity detection method of miRNA Download PDFInfo
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Abstract
Nucleic acid is efficiently conveyed the present invention provides DNA nanometers of tetrad realizations and the super sensitivity detection method of miRNA, the detection method include the following steps：(1) H1 nanometers of tetrads and H2 nanometers of tetrads are designed according to target miRNA, the H1 nanometers of tetrad and H2 nanometers of tetrads are combined by a Streptavidin molecule with four biotinylated fluorescence hairpin probe H1, H2 respectively to be formed；(2) Crosslinking Hybridization Chain Reaction (cHCR) react, and the initiation chain of cHCR reactions is miRNA；(3) external quantitative fluorescence analysis；(4) intracellular highly sensitive imaging analysis.Detection method nucleic acid transfer efficiency of the present invention is high, by causing cHCR reaction detection targets miRNA, the cHCR reactions that target miRNA specificity causes can form three-dimensional cross-linked hydrogel network, keep the reaction sensitivity high and the specificity detected is good, high selectivity provides high sensitivity and spatial resolution for intracellular imaging.
Description
Technical field
The invention belongs to biochemical fields, are efficiently conveyed to nucleic acid more particularly, to a kind of realization of DNA nanometers of tetrad
And the super sensitivity detection method of miRNA.
Background technology
Nucleic acid is in many basic cell processes, such as carrier of genetic information, intracellular regulatory molecules and catalyzing enzyme are all
It plays a crucial role.Because nucleic acid not only has the ability of standard base pairing, can also be carried out with various different moleculars
Conformation combines, it becomes cell biology and a kind of valuable adjusting and analysis work with unique specificity in medicine and pharmacology
Tool.Nucleic acid is applied to biomedical be mainly that can effectively be transported in different cells by transfection system.Closely
Nian Lai, DNA/RNA nanostructure as a kind of intracellular carrying method of very attractive nucleic acid, most it is representative just
It is spherical nucleic acid (SNAs) and self assembly nucleic acid nano structure.These nucleic acid nano structures do not need the auxiliary of cation carrier just
Cell can be entered, in living cells and internal explorative diagnostic analysis, curative drug conveying and Gene regulation provide
One powerful platform.However, in synthesizing and purifying, the efficiently conveying into cell and detection sensitivity etc. in the cell
These aspects, DNA/RNA nanostructures remain great challenge.
Protein provides a kind of potential timbering material for structure DNA/RNA nanostructures.With synthetic nanometer
Particle is different, protein surface uneven distribution active site, and protein core has explicit structure and difference
Oligomer structure.Based on these advantages, using albumen as core, the intensive work(of DNA is realized using its surface amine groups and sulfydryl
The SNAs of energyization has been devised.The SNAs of this specific type can not only promote cell to take the photograph functional protein
It takes, and basis can also be provided for the accurate nano-particle superlattice structure that adjusts.However, being received for this protein-nucleic acid
What rice structure studied in terms of the efficient conveying of synthesizing and purifying and nucleic acid almost without.Therefore, we construct one it is novel
The protein-nucleic acid nanostructure (DNA nanotetrads) being made of the hair clip DNA probe of SA and four biotin labeling,
It is conveyed for effective nucleic acid.Also, we combine HCR iodines, develop one kind and are used for based on crosslinking HCR amplification methods
The general-purpose platform of intracellular miRNA detections.
Invention content
In view of this, the present invention is directed to propose DNA nanometers of tetrad realizations are efficiently conveyed to nucleic acid and the hypersensitive of miRNA
Detection method, the transfer efficiency to solve intracellular nucleic acid and drug is low and miRNA detection sensitivities in the cell are low etc.
Problem.
In order to achieve the above objectives, the technical proposal of the invention is realized in this way：
DNA nanometers of tetrad realizations are efficiently conveyed to nucleic acid and the super sensitivity detection method of miRNA, the detection method include
Following steps：
(1) H1 nanometers of tetrads are designed according to target miRNA and H2 nanometers of tetrads, the H1 nanometers of tetrad and H2 receives
Rice tetrad, which is respectively combined by a Streptavidin molecule with four biotinylated fluorescence hairpin probe H1, H2, to be formed；
(2) HCR reacts, and the initiation chain of HCR reactions is miRNA；
(3) external quantitative fluorescence analysis；
(4) intracellular highly sensitive imaging analysis.
Further, H1 marks Cy3 as fluorogenic donor in the step (1), and H2 marks Cy5 as fluorescent receptor.
Further, the preparation method of described H1 nanometers of tetrads of step (1) and H2 nanometers of tetrads is as follows：Hairpin probe
H1 and H2 first carries out quenching pretreatment, respectively by final concentration of 2~9 μM of probe H1 and H2 under the conditions of 1 × PBS buffer solution 95
DEG C heating 2~10 minutes, be put into ice water 30 minutes, be then continuing at room temperature immediately after, be then added appropriate
SA and appropriate mixing H1 nanometers of tetrads of formation and H2 nanometers of tetrads.
Further, step (2) the HCR reactions are in the presence of having target miRNA, it opens hairpin probe H1, so
Afterwards by the way that alternate cycles hybridization is reacted cHCR occurs successively between probe H1 and H2, three-dimensional cross-linked hydrogel network is formed,
Cy3 fluorogenic donors and Cy5 receptors are closely coupled, and activation fluorescence resonance energy transfer signal does not occur in the absence of target
CHCR reacts, and does not generate fluorescence resonance energy transfer signal between Cy3 and Cy5.
Further, the analysis method of the step (3) is as follows：Include 1x XTAE-Mg in 30 μ L reaction systems2It is slow
H1 nanometer tetrads and H2 nanometers of tetrads that ultimate density is 110~180nM is added in fliud flushing, by a concentration of 0nM, 0.05nM,
The target miRNA-21 of 0.1nM, 0.25nM, 1nM, 2.5nM, 10nM, 25nM, 100nM cultivate 4h in 37 DEG C of constant temperature, add after reaction
Enter 70 μ L water and carry out fluoremetry, the measurement of fluorescence signal uses Fluorescence Spectrometer, excitation wavelength 540nm, exciting light and transmitting
Optical slits width is 5.0nm.
DNA nanometers of tetrad realizations are efficiently conveyed to nucleic acid and the super sensitivity detection method of miRNA is in tumour cell
Application in miRNA detections.
DNA nanometers of tetrad realizations are efficiently conveyed to nucleic acid and the super sensitivity detection method of miRNA is efficiently conveyed in nucleic acid
And the application in curative drug conveying.
Compared with the existing technology, DNA nanometers of tetrad realization of the present invention efficiently conveys nucleic acid and miRNA surpasses
Sensitive detection method has the advantage that：
(1) DNA nanometers of tetrad of the present invention realization efficiently conveys nucleic acid and the super sensitivity detection side of miRNA
Method, nucleic acid transfer efficiency is high, and by causing cHCR reaction detection target miRNA, the cHCR that target miRNA specificity causes reacts
Three-dimensional cross-linked hydrogel network can be formed, specific good, the high selectivity for keeping the reaction sensitivity high and detecting, is cell
Interior imaging provides high sensitivity and spatial resolution；
(2) accurate control of the DNA nanometers of tetrad of the present invention because of the simplicity of its synthesizing and purifying, structure and concentration
System, nucleic acid quick conveying and lysosome effective escape capability, be a kind of very powerful nucleic acid delivery of therapeutic agents, be nucleic acid and
Drug conveying opens new approach.
Description of the drawings
The attached drawing for constituting the part of the present invention is used to provide further understanding of the present invention, schematic reality of the invention
Example and its explanation are applied for explaining the present invention, is not constituted improper limitations of the present invention.In the accompanying drawings：
Fig. 1 is the principle of the present invention schematic diagram；
Fluorescence spectrum response diagram after the reaction of different component described in the embodiment of the present invention of the positions Fig. 2；
Fig. 3 is the agarose gel electrophoresis image described in the embodiment of the present invention
Wherein：Swimming lane 1：DNA Marker(50bp-10kb)；Swimming lane 2：H3+H4；Swimming lane 3：miRNA-21+H3+H4；Swimming
Road 4：H3nanotetrads+H4nanotetrads；Swimming lane 5：miRNA-21+H3nanotetrads+H4nanotetrads；
Fig. 4 is the copolymerization coke cell fluorescence imaging described in the embodiment of the present invention；
DNA nanometer tetrad sensitivity described in the embodiment of the present invention of the positions Fig. 5 and detection limit fluorescence spectra
Wherein：A) it is the miRNA detection sensitivities analysis based on DNA tetrads；(B) be DNA tetrads to not having to concentration
The response relation of miR-21；
Fig. 6 is the selective analysis chart of the DNA nanometer tetrads described in the embodiment of the present invention；
Fig. 7 is the intracellular miRNA-21 cell imagings figures of Hela described in the embodiment of the present invention
Wherein (A) is not processed, and inhibitor is added in (B), and simulation miR-21 is added in (C)
Specific implementation mode
With reference to embodiment and attached drawing, the present invention will be described in detail.
Embodiment 1
MiRNA is by taking miRNA-21 as an example for the embodiment target.
DNA nanometers of tetrad realizations are efficiently conveyed to nucleic acid and the super sensitivity detection method of miRNA, the detection method include
Following steps：
(1) H1 nanometers of tetrads are designed according to target miRNA and H2 nanometers of tetrads, the H1 nanometers of tetrad and H2 receives
Rice tetrad, which is respectively combined by a Streptavidin molecule with four biotinylated fluorescence hairpin probe H1, H2, to be formed,
H1 marks Cy3 as fluorogenic donor, and H2 marks Cy5 as fluorescent receptor
(2) cHCR reacts, and the initiation chain of cHCR reactions is miRNA；
(3) external quantitative fluorescence analysis；
(4) intracellular highly sensitive imaging analysis.
The preparation method of described H1 nanometers of tetrads of step (1) and H2 nanometers of tetrads is as follows：Hairpin probe H1 and H2 are first
Quenching pretreatment is carried out, final concentration of 7 μM of probe H1 and H2 are heated under the conditions of 1 × PBS buffer solution at 95 DEG C to 6 points respectively
Clock is put into ice water 30 minutes, is then continuing at room temperature immediately after, and SA appropriate and appropriate mixing shape is then added
At H1 nanometers of tetrads and H2 nanometers of tetrads.
Step (2) the HCR reactions are in the presence of having target miRNA, it opens hairpin probe H1, then by visiting
Alternate cycles hybridization is reacted cHCR occurs successively between needle H1 and H2, forms three-dimensional cross-linked hydrogel network, Cy3 fluorogenic donors
It is closely coupled with Cy5 receptors, fluorescence resonance energy transfer signal is activated, in the absence of target, cHCR reactions, Cy3 do not occur
Fluorescence resonance energy transfer signal is not generated between Cy5.
The analysis method of the step (3) is as follows：Include 1x TAE-Mg in 30 μ L reaction systems2Buffer solution is added most
The H1 nanometer tetrads and H2 nanometers of tetrads of final concentration of 150nM, by a concentration of 0nM, 0.05nM, 0.1nM, 0.25nM,
The target miRNA-21 of 1nM, 2.5nM, 10nM, 25nM, 100nM cultivate 4h in 37 DEG C of constant temperature, and the progress of 70 μ L water is added after reaction
The measurement of fluoremetry, fluorescence signal uses Fluorescence Spectrometer, and excitation wavelength 540nm, exciting light and transmitting optical slits width are equal
For 5.0nm, fluorogram is shown in Fig. 2.
Agarose gel electrophoresis is analyzed
To verify the structure of nano-particle, We conducted agarose gel electrophoresis analyses.20 μ L1 μM SA's of every pipe
In 1XPBS solution, the H3 of different solubility is added, forms four kinds of SA-DNA compounds, different samples is made.Glue and electrophoresis mistake
Cheng Jun is carried out at room temperature, Ago-Gel and 0.5 × tbe buffer liquid (45mM Tris, 45mM of the electrophoretic analysis using 3%
Boric Acid, 10mM EDTA, pH 8.0), it is dyed with 5 μ L GelRED.10 μ L sample mixed liquors are added in well,
Electrophoresis 90 minutes under 110V voltages.After electrophoresis, observes band with 240 gel imaging systems of tocan and take pictures.
With the relevant electrophoresis experiment samples of HCR in 20uL1XTAE-Mg2 buffer solutions (10mm Tris, 12.5mm
MgCl2, pH 7.6) in 37 DEG C incubation 6h.Hcr products are analyzed with 2% Ago-Gel.Gel 0.5g/mL
Goldview and 0.5g/mL ethidium bromide stainings.10 μ L samples are added in well, the electrophoresis 2h under the constant voltage of 100V.
After electrophoresis, observes band with 240 gel imaging systems of tocan and take pictures, concrete outcome is shown in Fig. 3.
The application of DNA nanometers of tetrads biological imaging in the cell
We first study the property of intracellular DNA nanometers of tetrad, we are in synthetic DNA nanometer tetrad
When selection using label Cy3 SA or mark Cy5 probe H2.After cell incubation 1h, as a result as shown in figs. 4 b-c, cell cytosol
Interior is in bright fluorescence.And incubated cell is removed with the SA of free label Cy3 in check experiment, apparent fluorescence is not observed,
As shown in Figure 4 A, this is the result shows that DNA nanometers of tetrads can enter cell in the case where that need not transfect.According to this
Experimental result, clearly show that protein surface combine DNA probe be clearly DNA nanometers of tetrads enter cell it is crucial because
Element.For further research, cell is incubated with the DNA tetrads of Cy3 and Cy5 with being marked simultaneously.As a result
It has been shown that, in cell Cy3 and Cy5 fluorescence channels have strongly with the fluorescence signal of height.DNA nanometers of tetrads are incubated with SA
The albumen for further inquiring into Cy3 the and H2 probes label marked with Cy5 of cell is all shown in the intracellular of the channels Cy3 and Cy5
Go out the common location fluorescence signal strongly with height, such as Fig. 4 D-F, this discovery demonstrates this DNA nanometers of tetrad simultaneously
The property for the structural intergrity that can't be detached from from SA albumen with biotinylated DNA probe in the cell.
Vitro detection and selectivity based on DNA nanometers of tetrad sensors
We choose the change intensity of FRET signals to investigate the detection performance of sensor.Fig. 5 A describe to be based on DNA
Fluorescent spectrum curve of the tetrad nano-sensor under different target miRNA-21 concentration.As seen from the figure, FRET signals are strong
Degree is gradually increased with aimed concn in 5pM to 100nM ranges.The phenomenon illustrates, with opening wide for aimed concn, initiation
HCR reactions are more, and the FRET signals of generation are stronger.Fig. 5 B are the fluorescence intensity (F of fluorescent receptorA) and the fluorescence of fluorogenic donor it is strong
Spend (FD) ratio of fluorescence intensity and various concentration miRNA-21 correction graphs i.e. at 662nm/562nm.According to 3 σ rules
The detection being calculated is limited to 6pM, is better than the sensitivity of existing miRNA-21 imaging methods.The above results show this experiment side
Method can be used for the highly sensitive detection of miRNA-21.
To investigate the application potential of this sensor, our selectivity to it and the target sample in complex biological sample
Detection performance is assessed.By Fig. 6 the result shows that, using other miRNA, β-actin or negative control cell L-02
Lysate when, the FTER signals of sensor with change when blank sample almost without or variation have small, illustrate to miRNA-
21 have good selectivity.The experimental results showed that the sensor still has significantly the target miRNA in complex biological environment
Response, it was demonstrated that this method has the potentiality of practical application.
Intracellular miRNA-21 cell imagings
On the basis of the above experimental result, we are imaged using DNA nanometers of tetrad sensors in living cells.In Fig. 7 A
Double-colored confocal images are shown, as HeLa cells two kinds of DNA nanotetrads, i.e. H1 nanotetrads and H2
After nanotetrads is incubated simultaneously, apparent green channel fluorescence and red channel fluorescence can be observed simultaneously in cell, it is green
Color fluorescence channel represents Cy3 fluorescence signals, and red fluorescence channel represents FRET fluorescence signals.This illustrates to have caused cHCR anti-
It answers, to produce FRET signals.In order to further prove the specificity of fluorescence signal, we are transfected with transfection agents first
(can the specifically bind miRNA-21 and selectivity of design reduce the synthesis list of miRNA-21 active concentrations to miRNA-21 inhibitor
Chain RNA) enter cell, as result shows that the enhancing of green fluorescence, and the decrease of red fluorescence show that miRNA-21 draws in Fig. 7 B
The reduction of the cHCR reactions of hair.On the contrary, if cell transfects the miRNA-21 analog chains of synthesis in advance improves Hela to reach simulation
Intracellular miRNA-21 expressions, compared with not pretreated HeLa cells, red English light is stronger and green fluorescence is opposite
Weaken (Fig. 7 C).These results indicate that fluorescence signal is in dynamical correlation with miR-21 concentration, the hair of cHCR reactions is directly demonstrated
It is raw, and spurious signal is can avoid to distinguish different intracellular miRNA expressions.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention god.
Sequence table
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Claims (7)
1.DNA nanometers of tetrad realizations are efficiently conveyed to nucleic acid and the super sensitivity detection method of miRNA, it is characterised in that：The inspection
Survey method includes the following steps：
(1) H1 nanometers of tetrads and H2 nanometers of tetrads, the H1 nanometers of tetrad and H2 nanometers four are designed according to target miRNA
Fission, which is respectively combined by a Streptavidin molecule with four biotinylated fluorescence hairpin probe H1, H2, to be formed；
(2) HCR reacts, and the initiation chain of HCR reactions is miRNA；
(3) external quantitative fluorescence analysis；
(4) intracellular highly sensitive imaging analysis.
2. DNA nanometers of tetrad realization according to claim 1 is efficiently conveyed to nucleic acid and the super sensitivity detection side of miRNA
Method, it is characterised in that：H1 marks Cy3 as fluorogenic donor in the step (1), and H2 marks Cy5 as fluorescent receptor.
3. DNA nanometers of tetrad realization according to claim 1 is efficiently conveyed to nucleic acid and the super sensitivity detection side of miRNA
Method, it is characterised in that：The preparation method of described H1 nanometers of tetrads of step (1) and H2 nanometers of tetrads is as follows：Hairpin probe H1
First carry out quenching pretreatment with H2, respectively by final concentration of 2~9 μM of probe H1 and H2 under the conditions of 1 × PBS buffer solution at 95 DEG C
Heating 2~10 minutes, is put into ice water 30 minutes, is then continuing at room temperature, SA appropriate is then added immediately after
And appropriate mixing forms H1 nanometers of tetrads and H2 nanometers of tetrads.
4. DNA nanometers of tetrad realization according to claim 1 is efficiently conveyed to nucleic acid and the super sensitivity detection side of miRNA
Method, it is characterised in that：Step (2) the cHCR reactions are in the presence of having target miRNA, it opens hairpin probe H1, then
By the way that alternate cycles hybridization is reacted HCR occurs successively between probe H1 and H2, three-dimensional cross-linked hydrogel network, Cy3 are formed
Fluorogenic donor and Cy5 receptors are closely coupled, and in the absence of target cHCR does not occur for activation fluorescence resonance energy transfer signal
Reaction, does not generate fluorescence resonance energy transfer signal between Cy3 and Cy5.
5. DNA nanometers of tetrad realization according to claim 1 is efficiently conveyed to nucleic acid and the super sensitivity detection side of miRNA
Method, it is characterised in that：The analysis method of the step (3) is as follows：Include 1x TAE-Mg in 30 μ L reaction systems2Buffer solution,
The H1 nanometer tetrads and H2 nanometers of tetrads that ultimate density is 110~180nM is added, by a concentration of 0nM, 0.05nM,
The target miRNA-21 of 0.1nM, 0.25nM, 1nM, 2.5nM, 10nM, 25nM, 100nM cultivate 4h in 37 DEG C of constant temperature, add after reaction
Enter 70 μ L water and carry out fluoremetry, the measurement of fluorescence signal uses Fluorescence Spectrometer, excitation wavelength 540nm, exciting light and transmitting
Optical slits width is 5.0nm.
6. the DNA nanometer tetrads realization as described in claim 1-5 is efficiently conveyed to nucleic acid and the super sensitivity detection side of miRNA
Application of the method in tumour cell in miRNA detections.
7. the DNA nanometer tetrads realization as described in claim 1-5 is efficiently conveyed to nucleic acid and the super sensitivity detection side of miRNA
Application of the method in nucleic acid efficiently conveys and curative drug conveys.
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|---|---|---|---|---|
| CN109540856A (en) * | 2018-11-08 | 2019-03-29 | 南京师范大学 | A kind of reagent based on fluorescence resonance energy transfer detection different subtype breast cancer cell |
| CN109781688A (en) * | 2019-01-18 | 2019-05-21 | 青岛科技大学 | The method that the construction method and Ratio-type of DNA silicon nano-hydrogel based on Nano particles of silicon dioxide detect intracellular ATP |
| CN111549102A (en) * | 2020-05-28 | 2020-08-18 | 西南大学 | Amphiphilic DNA nano micelle and preparation method and application thereof |
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| CN113736856A (en) * | 2021-09-27 | 2021-12-03 | 南开大学 | Isothermal nucleic acid amplification sensor for rapidly detecting hypochlorous acid and myeloperoxidase |
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