CN107988351A - A kind of application of cyclic DNA in the detection, imaging and gene therapy of just RNA - Google Patents
A kind of application of cyclic DNA in the detection, imaging and gene therapy of just RNA Download PDFInfo
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Abstract
The present invention provides a kind of application of cyclic DNA in the detection, imaging and gene therapy of just RNA, the cyclic DNA includes at least one or more DNA probe fragments with detection target justice RNA sequence complementation, and the cyclic DNA is formed by 3 ' and 5 ' the end connection cyclisation of ssDNA itself.By making cyclic DNA form heteroduplex with detection target justice RNA, heteroduplex is set to depart from from carrier, so that（1）Realize that detection, the application of imaging are realized in corresponding fluorescence signal change；（2）It is enzymatically decomposed the just RNA in heteroduplex, so as to fulfill gene therapy purpose.
Description
Technical field
The present invention relates to application of the cyclic DNA in detection, imaging and field of gene, more particularly to a kind of ring-type
DNA detected in just RNA, be imaged and gene therapy in application, belong to biological technical field.
Background technology
DNA is considered as the ideal structure for building molecular probe in recent years in addition to the inhereditary material as organism
Unit, and bioanalysis and biomedicine field are widely used in, it is more including aptamer, molecular beacon and DNAzyme
Kind functionalization DNA is used for bio-sensing, diagnosis and disease treatment.DNA has that synthesis is simple, is appropriate for structural modification, choosing
The advantages that selecting property and high compatibility, but the maximum obstruction that DNA is applied to be faced in biological living at the same time is stability and biology
Utilization rate is not high enough, thus can produce since false positive signal or therapeutic effect are paid no attention to caused by albumen interference and enzyme degraded
Think.Therefore, many methods all be used to overcome these problems.
According to the research of early stage, nucleic acid derivative by different chemical modifications synthesis is due to can not be by endogenic nucleic acid
Decomposition mechanism identifies, such as thiophosphorylation modification, lock nucleic acid substituent, 2 '-glycosyl site are carried out on the phosphate backbones of DNA
Substituted by methoxyl group and fluorine.In addition to these method of modifying, cyclisation also provides another kind for raising Nucleic acid stabilization can
Can property.
The nineties in last century, it has been found that except wire nucleic acid also has another nucleic acid --- circular rna in life entity
（circRNA）.In recent years, numerous studies begin to focus on endogenous circRNA, these researchs are focused primarily upon and will utilized
CircRNA is adjusted the activity of related miRNA or albumen by sponge regulation mechanism.Due to being lacked in circRNA structures
Free end less, it has good stability, and half-life period from a few houres in vivo were differed by several days.In addition, and its
He compares chemical modification mode, and the cyclisation of nucleic acid only needs natural nucleotide to participate in, and avoiding can due to introducing unnatural nucleic acids
The genotoxic potential that can be brought.
Therefore, some methods for being used for external synthesis of cyclic nucleic acid are developed in recent years, they are applicable not only to RNA cyclisation
Also cyclisation for DNA is expanded.In view of the plurality of advantages of circularized nucleic acid, a kind of simple cyclic DNA is constructed in of the invention
Probe is used for biological living.Although having had the more synthesis on cyclic DNA and property Quality Research at present, have no it
Detection, imaging and gene therapy applied to live body.
The content of the invention
The present invention provides a kind of application of cyclic DNA in the detection, imaging and gene therapy of just RNA, the ring
Shape DNA includes at least one or more DNA probe fragments with detection target justice RNA sequence complementation, the cyclic DNA by
3 ' and 5 ' the end connection cyclisation of ssDNA itself are formed.By making cyclic DNA form heteroduplex with detection target justice RNA, from
And（1）Realize that detection, the application of imaging are realized in corresponding fluorescence signal change；（2）Heteroduplex is departed from from carrier, make just
Adopted RNA is enzymatically decomposed, so as to fulfill the application of gene therapy.
Circular DNA probes are used for the detection of justice RNA by the present invention first；Further, circular DNA probes are used for thin
The detection and gene therapy of intracellular justice RNA；Further, by the nucleic acid of the similar performance such as cyclic DNA and graphene oxide
Carrier combines the detection and gene therapy that structure bioanalytical sensing platform is used for justice RNA in living cells.
Concrete technical scheme provided by the invention is as follows：
In a first aspect, the application the present invention provides a kind of cyclic DNA in the detection or imaging of just RNA, specifically, described
Cyclic DNA includes one or more and detection target justice RNA sequence complementation DNA probe fragment, fluorescent marker；The ring
Shape DNA is formed by 3 ' and 5 ' the end connection cyclisation of ssDNA itself.
Specifically, cyclic DNA is as follows by the process that ssDNA itself connections cyclisation forms：（1）Add primer sequence and be used as and draw
Dosage form is sent out into after local duplex structure, is reacted under the catalysis of T4 DNA ligases；（2）Heating inactivates ligase；（3）Add outer
Unreacted ssDNA and primer sequence are degraded in enzyme cutting；（4）Heating inactivates excision enzyme；（5）Product purification, obtains pure
Cyclic DNA, can be quantified by the absorbance that ultraviolet-visible spectrophotometer is measured at 260nm as needed.
The present invention uses general fluorescent marker, such as：The fluorescein isothiocynate of fluoresceins（FITC）, hydroxyl it is glimmering
Light element（FAM）, tetrachlorofluorescein（TET）Deng；Cy3, Cy5 of cyanine dyes class etc.；And luxuriant and rich with fragrance ingot class, rhodamine, quantum dot class
Deng fluorescent marker.
Further, this method comprises the following steps：（1）The cyclic DNA is compounded to form with fluorescence quenching compound
Probe；（2）Make the combined probe with comprising detection target justice RNA thing to be detected interact, formed cyclic DNA with just
The heteroduplex of adopted RNA；（3）Detect the fluorescence intensity of the cyclic DNA and the heteroduplex of justice RNA.
Further, the fluorescence quenching has fluorescence quenching capability, and is higher than with the binding ability of single-chain nucleic acid
With the binding ability of double-strandednucleic acid.As preference, the fluorescence quenching is graphene oxide, carbon nanotubes, carbonitride are received
Rice piece, manganese dioxide nano-plates or boron nitride nanosheet.Specifically, such as graphene oxide（GO）It is that one kind has planar structure
And the two-dimension nano materials of many properties.Since graphene oxide has high load amount, quenching intensity and cell transport
Ability, is widely used in and the common constructing function platform of nucleic acid, such as molecular probe, cell imaging and medicine delivery
Deng.
Further, to improve detection efficiency, the cyclic DNA includes more than two with detecting target justice RNA sequences
Complementary DNA probe fragment is arranged, further includes the junction fragment for connecting each DNA probe fragment.
Further, as general selection, it is mRNA, siRNA or microRNA to tell just RNA.
Further, the present invention realizes vivo detection, is specifically, step（2）Described in comprising detection target justice
The thing to be detected of RNA is cell, and the combined probe is incubated jointly with cell, forms cyclic DNA and justice RNA's in the cell
Compound.
Second aspect, the present invention provides a kind of application of cyclic DNA in the gene therapy of just RNA, including it is as follows
Step：The nucleic acid carrier of the load cyclic DNA is set to enter with the cell comprising detection target justice RNA, forming cyclic DNA
With the heteroduplex of just RNA and departing from from the nucleic acid carrier, the just RNA in the heteroduplex is enzymatically decomposed, phase
The expression process of correlation gene is obstructed；The cyclic DNA includes one or more DNA with detection target justice RNA sequence complementation
Probe fragment；The cyclic DNA is formed by 3 ' and 5 ' the end connection cyclisation of ssDNA itself.
Further, to improve gene therapy effect, the cyclic DNA includes more than two just with detection target
The DNA probe fragment of RNA sequence complementation, further includes the junction fragment for connecting each DNA probe fragment.
Further, the justice RNA is mRNA, siRNA or microRNA；The nucleic acid carrier for graphene oxide,
Carbon nanotubes, azotized carbon nano piece, manganese dioxide nano-plates or boron nitride nanosheet.Specifically, graphene oxide（GO）It is one
Two-dimension nano materials of the kind with planar structure and many properties.Since graphene oxide has high load amount, quenching
Ability and cell transport capacity, are widely used in and the common constructing function platform of nucleic acid, such as molecular probe, cell imaging
And medicine delivery etc..
Technical scheme is at least up to following beneficial effect compared with the prior art：
（1）Since there is cyclic DNA many to be better than single stranded DNA（ssDNA）Property, such as stronger antienzyme cut ability and
More efficient, stability is identified to intracellular antisense RNA using cyclic DNA in stability in biofluid, the present invention
More preferably.
（2）Introducing has a fluorescence quenching capability, and with the binding ability of single-chain nucleic acid higher than and double-strandednucleic acid combination
The fluorescence quenching of ability, as graphene oxide is for composite with cyclic DNA, realizes and is efficiently imaged in living cells
With treatment since cyclic DNA lacks free-end, it is not easy to be degraded by exonuclease, and the parent of cyclic DNA and fluorescence quenching
Higher with property, cyclic DNA is not likely to produce in living cells or in vivo is produced non-specific inspection by nuclease and albumen interference
Signal is surveyed, there is the signal-to-noise ratio of higher and relatively low false positive signal.
（3）Since cyclic DNA lacks free-end, it is not easy to be degraded by exonuclease, therefore, cyclic DNA is in living cells
Or in vivo the holdup time is longer, for gene therapy when is more efficient.
（4）Nucleic acid probe compound can enter intracellular acidity and rich in big after cell is entered by endocytic pathway
In the vesica for measuring enzyme, including early stage endosome, late period endosome and lysosome.Since cyclic DNA has the stability of higher, energy
It is not degraded in endosome/lysosome, finally escape into cytoplasm, performance detection is combined with target justice RNA
With the function of gene therapy.
（5）Cyclic DNA can more effectively suppress the translation of just RNA, so that the more effectively table of suppression target albumen
Reach and then suppress the propagation of cell.
Therefore, cyclic DNA can be used as efficient biological detection, sensing and treatment tool in biological living.
Brief description of the drawings
Fig. 1 is cyclic DNA and the application in corresponding ssDNA justice RNA detections in the cell, imaging and gene therapy
Schematic diagram.
Fig. 2 is（a）The gel electrophoresis figure of cyclic DNA and ssDNA；（b）Circular DNA and ssDNA are incubated not in containing 10% serum
With the gel electrophoresis figure of product after the time.
The FP values of Fig. 3 a Cy5- cyclic DNAs and Cy5-ssDNA in different solutions environment.
Fluorescence emission spectrums of Fig. 3 b Cy5- cyclic DNAs/GO in various concentrations target mRNA solution.
Fig. 3 c Cy5- cyclic DNAs/GO and the Cy5-ssDNA/GO fluorescence kinetic profiles in 10%FBS solution.
Fig. 3 d Cy5- cyclic DNAs/GO and Cy5-ssDNA/GO with complete complementary desired mRNA sequences（Solid line）Or single alkali
Base mutant target mRNA sequence（Dotted line）Fluorescence kinetic profiles after being incubated altogether.
Fig. 4 is（a）Lg (the F that cyclic DNA and ssDNA are combined with GO0/F-1)-lgCGOCurve；（b）Cyclic DNA/GO and
Fluorescence kinetic profiles of the ssDNA/GO when producing response to target mRNA；（c）Cy5- cyclic DNAs and Cy5-ssDNA be not by
With the ratio of concentration GO quenching fluorescences；（d）Cy5- cyclic DNAs/GO and Cy5-ssDNA/GO targets in 10% serum solution is detected
The signal-to-background ratio S/B of sequence.
Fig. 5 is HeLa cells and survivin mRNA targetings（a）Cy3- cyclic DNAs/GO or（b）Cy3-ssDNA/GO
The laser co-focusing image after different time is incubated altogether.
Fig. 6 is MCF-10A cells and the cyclic DNA/GO or ssDNA/ of survivin mRNA and c-raf mRNA targetings
GO is incubated the laser co-focusing image after 60min altogether.
Fig. 7 a are cyclic DNA/GO in Subcellular Localization Confocal Images intracellular HeLa.
Fig. 7 b ssDNA/GO are in Subcellular Localization Confocal Images intracellular HeLa.
Fig. 8 is（a）HeLa cells are incubated the cell survival after 24h altogether with various concentrations cyclic DNA/GO or ssDNA/GO
Rate；After HeLa cells are incubated 24h altogether with various concentrations cyclic DNA/GO or ssDNA/GO（b）Survivin and（c）C-raf kinases
Expressing quantity.
Fig. 9 is incubated the cell survival after 24h altogether for MCF-10A cells with various concentrations cyclic DNA/GO or ssDNA/GO
Rate.
Embodiment
Hereinafter, the specific implementation example of the present invention, skill of the invention are explained in more detail using embodiment and comparative example
Art scope is not limited to following embodiments.
Intuitively to show the technology contents of the embodiment of the present invention and comparative example, description below is done referring to Fig. 1：
As shown in Figure 1, the embodiment of the present invention（Left part in Fig. 1）Middle cyclic DNA contains the sequence with the RNA complementations of target justice
Row, in the presence of target justice RNA, form cyclic DNA/justice RNA double stranded heteroduplex thing and from carrier（Using graphene oxide GO as
Example, as fluorescence quenching and nucleic acid carrier）On come off.With ssDNA（Right part in Fig. 1）Unlike, cyclic DNA has
Stronger stability being capable of resistant cells inclusion body and the degraded of intracytoplasmic enzyme.Therefore, cyclic DNA/GO has in the cell
The efficiency and performance having had.The present invention has not only inquired into the destiny of cyclic DNA/GO in the cell, at the same just RNA be imaged with
And the efficiency in terms of gene therapy is also studied.
Experimental article and associated verification method involved in present embodiment is as follows：
（1）Chemical reagent：All nucleic acid（Sequence is shown in Table 1）By raw work bioengineering（Shanghai）Limited company synthesizes and passes through
HPLC is purified.GO aqueous dispersions are purchased from Beijing nano and hold high Nono-material Science ＆ Technology Ltd..LysoTracker Green and
Hoechst 33342 is purchased from Thermo Fischer Scient Inc. of the U.S..T4 DNA ligases are purchased from NEB (Beijing) Co., Ltd.Outside
Enzyme cutting I and III is purchased from precious bioengineering（Dalian）Co., Ltd.Ultra-pure water is prepared by Milli-Q water purification systems（18.2MΩ）.
（2）Cell line and cell culture：People epithelium of cervix uteri cancer cell HeLa and normal human mammary epithelial cell MCF-10A purchases
In American Type Culture Collection（ATCC）.HeLa cell culture is in 1640 culture mediums of RMPI（HyClone）Add 10% tire
Cow's serum（Gibco）With 100 IU/mL Pen .- Streps, contain 5% CO at 37 DEG C2Atmosphere in cultivated.MCF-10A
Cell growth is in the galactophore epithelial cell culture medium MEGM kit containing cholera toxin（Lonza）, contain 5% CO at 37 DEG C2Gas
Cultivated in atmosphere.
Embodiment 1
The preparation of cyclic DNA：Formed by ssDNA itself connection cyclisation, add primer sequence and form local pair as initiator
After chain structure, overnight, 10min is then placed at 65 DEG C inactivates ligase for 16 DEG C of reactions under the catalysis of T4 DNA ligases.
It is subsequently added into exon Ⅰ and III unreacted ssDNA and primer sequence degrade in 37 DEG C of reaction 1h, is heated to 90 DEG C of holdings
10min inactivates enzyme.Finally, with II gel reclaims kits of QIAEX（Qiagen）Product is purified, is obtained pure
Cyclic DNA, the absorbance measured by ultraviolet-visible spectrophotometer at 260nm are quantified.
Using Survivin mRNA as target justice RNA in the present embodiment 1, the sequence ginseng of corresponding ssDNA and justice RNA
It is shown in Table 1.
Comparative example 1
Cyclic DNA, remaining experiment condition all same are directly substituted with the ssDNA in embodiment 1.
The sequence table of the ssDNA and justice RNA that are used in 1 embodiment 1 of table and comparative example 1
Note：The target sequence of survivin probes is the NO.304-NO.323 for the survivin mRNA that total length is 2655-bp
Base section（Numbering is NM001168 in GenBank）
Embodiment 2
Usingc-raf MRNA is as target justice RNA, and the preparation of corresponding cyclic DNA is with embodiment 1, corresponding ssDNA and just
The sequence of adopted RNA is referring to table 2.
Comparative example 2
Cyclic DNA, remaining experiment condition all same are directly substituted with the ssDNA in embodiment 2.
The sequence table of the ssDNA and justice RNA that are used in 2 embodiment 2 of table and comparative example 2
Note：c-rafThe target sequence of probe is that total length is 3282-bpc-raf The NO.2771-NO.2790 alkali of mRNA
Between base（Numbering is NM002880 in GenBank）
（One）Related experiment process
Related cyclic DNA in embodiment 1, comparative example 1, embodiment 2, comparative example 2, ssDNA are subjected to following related experiment：
A. DNA stability is evaluated in serum：
2 μM of cyclic DNAs are prepared with the RPMI 1640 containing 10% hyclone or ssDNA solution is placed at 37 DEG C and is incubated.When each
Between point be sampled, 5min are heated at 95 DEG C makes enzyme inactivation in serum, and -80 DEG C of sample saves backup.
B. polyacrylamide gel electrophoresis（PAGE）Analysis：
The heterogeneity of cyclic DNA is analyzed with urea-denatured PAGE and Native PAGE respectively.The former is used to identify ring-type
Whether DNA successfully synthesizes, and the latter is used to verify the binding ability of cyclic DNA and target mRNA and the stability of DNA.
C. the fluorescence experiments in solution：
The cyclic DNA or ssDNA-2 of Cy5 marks are used to carry out spectrofluorimetry experiment, and the excitation light wave of use is a length of
640nm, collection wavelength are located at the emission spectrum in the range of 650-750nm.
D. confocal microscope is analyzed：
Seed cells into the burnt ware of 35mm copolymerization after cultivating 24h, add fluorescent marker cyclic DNA/graphene oxide GO or
SsDNA/ graphene oxides GO.By the common incubation in 37 DEG C of different times, with LysoTracker Green and Hochest
3324 are dyed, and fluorescence co-focusing imaging is carried out after cleaning.
E. cytotoxicity analysis：
The gene therapy effect of cyclic DNA/graphene oxide GO or ssDNA/ graphene oxide GO is analyzed by CCK-8.
Absorbance is measured with microplate reader, calculates cell survival rate.
F. western blot analysis：
After HeLa cell culture 24h, add cyclic DNA/graphene oxide GO or ssDNA/ graphene oxide GO and be used for cell
Gene regulation.After being incubated 24h altogether, cell is collected and adds cell lysis buffer solution and protease inhibitors to extract the complete of cell
Albumen.Obtained protein extract is quantified with BCA protein quantification kits.Protein extract is taken to be mixed with sample-loading buffer
Electrophoresis is carried out in SDS-PAGE afterwards, is subsequently transferred to polyvinylidene fluoride（PVDF）On film.Film is dipped into containing confining liquid
Plate in close.Film is immersed in the solution of the primary antibody anti-survivin or anti-raf1 containing target protein, 4
DEG C be incubated overnight.Film is dipped into the solution containing the secondary antibody for being marked with HRP again, after 4 DEG C are incubated, adds HRP detection reagents
Carry out chemiluminescence imaging.
（Two）Related experiment result
A. the property of cyclic DNA：
It has selected mesh of the mRNA of 2 kinds of albumen as detection and treatment in embodiment 1, comparative example 1, embodiment 2, comparative example 2
Mark --- survivin and c-raf kinases, two kinds of albumen high expression in kinds of tumor cells, can be as the biological marker of tumour
Molecule.
3 sections of DNA sequence dnas with the RNA complementations of target justice are contained in the sequence of the cyclic DNA of synthesis, in target justice
Complementary hybridization occurs in the presence of RNA and forms cyclic DNA/justice RNA heterocomplexs.By gel electrophoresis verify cyclic DNA synthesis into
, there is the ssDNA that molecular weight is higher than same sequence in work(, DNA migration rate reductions in gel after cyclisation（Fig. 2 a）.Separately
Outside, stability of the cyclic DNA in serum is tested also by electrophoresis（Fig. 2 b）, it can be seen that ssDNA is containing tire ox blood
It is completely degraded in clear culture medium after 3h, but just there is obvious degradation after 8h in cyclic DNA, therefore cyclic DNA is in life
There is the resistance to enzymolysis ability of higher in objects system.
In order to build sensing platform, it is necessary to be investigated to the interaction strength between cyclic DNA and graphene oxide GO.
Following experiments are carried out using the cyclic DNA of fluorescence molecule mark.First, fluorescence polarization is passed through（FP）To the knot of cyclic DNA and GO
Conjunction is verified that fluorescence polarization reflection is that fluorescence molecule is caused molecule turning power to change by residing microenvironment.
As shown in Figure 3a, the FP values of Cy5- cyclic DNAs are very low, and after GO is added, the increase of FP values, illustrates the absorption of Cy5- cyclic DNAs in GO
Surface forms compound.Add target justice RNA, FP value and be again lowered to the water close with the initial value of Cy5- cyclic DNAs
It is flat.This change procedure illustrates in the presence of target justice RNA, cyclic DNA and target justice RNA formed cyclic DNA/
Just RNA heterocomplexs simultaneously depart from from GO surfaces.Secondly, combined by measuring binding kinetics to evaluate cyclic DNA with ssDNA
The difference of compatibility during GO.According to 1 binding constant of formulaK A
Formula 1
In formula, F0With F be respectively nothing and in the presence of having GO Cy5- cyclic DNAs fluorescence intensity.As shown in fig. 4 a, it is dense in low GO
In the range of degree（0-0.8μg·mL-1）,K A Numerical value be equal to slope of a curve.The KA values of cyclic DNA/GO under the same conditions
（1.212 mL·μg-1）Higher than ssDNA/GO（0.060 mL·μg-1）, illustrate that the affinity that cyclic DNA is combined with GO is better than
ssDNA.In addition, in the presence of target justice RNA, fluorescence-time graph of cyclic DNA/GO and ssDNA/GO are also anti-
Mirror similar conclusion（Such as Fig. 4 b）.When, there are during target justice RNA, the speed that cyclic DNA departs from from GO is low in solution
In ssDNA.Drawn according to above-mentioned experimental data compared to ssDNA, the binding affinity of cyclic DNA and GO are stronger.
B. mRNA is detected in solution：
Cyclic DNA is combined to the bioanalytical sensing platform of structure identification target justice RNA with GO, investigates it in solution system first
Performance, including sensitivity, selectivity and antijamming capability.First, the dosage of GO is optimized, configured from 1 μ g/mL to 4 μ g/
The GO solution of mL various concentrations adds the combination buffer containing 100nM cyclic DNAs, and 665nm is measured after 37 DEG C of incubation 10min
The fluorescence intensity at place, the optimal concentration that GO is obtained according to measure curve is 3 μ g/mL（Such as Fig. 4 c）.Secondly, it is detected the survey of limit
Fixed, first Cy5- cyclic DNAs/GO or Cy5-ssDNA/GO compounds obtained in combination buffer, concentration is added into solution
For the target justice RNA of 1 nM to 1000 nM, after 37 DEG C are incubated, fluorescence emission spectrum is measured.Solution fluorescence intensity find with
The increase fluorescence intensity of justice RNA concentration progressively strengthens, and within the specific limits, fluorescence intensity and the concentration of justice RNA
It is proportional change（Fig. 3 b）.It is reachable to the test limit of target justice RNA according to the standard of three times signal-to-noise ratio, cyclic DNA/GO
To the test limit of 0.4nM, slightly below ssDNA/GO.What DNA/RNA associated proteins and endogenous nucleic acid enzyme in organism produced
Nonspecific interference is the application for limiting DNA biosensor in biological living.In order to solve this problem, cyclic DNA/GO
Detectability in complicated solution environment is another important evaluation criterion.By contrasting cyclic DNA/GO and ssDNA/GO
The dynamic changing curve of fluorescence in the solution containing 10% FBS（Such as Fig. 3 c）It can be found that ssDNA/GO occurs clearly
Fluorescence signal, and the fluorescent value of cyclic DNA/GO is held essentially constant, and illustrates that ssDNA/GO can be produced in complicated solution environment
Raw stronger false positive signal, cyclic DNA/GO more stablizes in biosystem is nearly free from nonspecific response letter
Number.Then, continue to investigate target detection capabilities of the cyclic DNA/GO in serum.Cy5- cyclic DNAs/GO or Cy5-
SsDNA/GO is used to detect the target justice RNA being dissolved in the buffer solution containing 10% serum, after being incubated 30min, record transmitting light
As shown in figure 4d, the background fluorescence signal of Cy5- cyclic DNAs/GO is significantly lower than Cy5-ssDNA/GO to spectrum, when target justice RNA is deposited
When, the fluorescence of Cy5- cyclic DNAs/GO is remarkably reinforced, and the Fluorescence Increasing of Cy5-ssDNA/GO is weaker, illustrates cyclic DNA/GO
With compared with the stronger antijamming capabilities of ssDNA/GO.Finally, in order to investigate the specificity of cyclic DNA/GO identification targets, use respectively
Cy5- cyclic DNAs/GO and Cy5-ssDNA/GO is to the target justice RNA or the just RNA of single base mismatch in solution（1-
mut）It is detected.After adding the target justice RNA of 300nM complete complementaries or the just RNA sequence of single base mismatch, monitoring
Fluorescence intensity change in 2h obtains kinetic curve.As shown in Figure 3d, cyclic DNA/GO has stronger mesh than ssDNA/GO
Mark selectivity.
In addition, introduce selectivity factor α carries out quantitative analysis to the selectivity of detection,, whereinOrRepresent probeThere are target in the solutionOrWhen signal-to-background ratio numerical value, define probe to completely mutually
The selectivity of the target justice RNA of complementary series is standard value, i.e.,.Cyclic DNA/GO and ssDNA/GO are to single base mismatch
The selectivity factor of just RNA sequence is respectively 0.313 and 0.745, illustrates identification energy of the cyclic DNA/GO to single base mismatch
Power is better than ssDNA/GO.To sum up, since cyclic DNA has the advantages that resistance to enzymolysis, high with GO compatibilities, cyclic DNA/GO pairs is utilized
Target justice RNA is detected the sensitivity that can obtain higher, lower background signal and better choice.
C. intracellular justice RNA imagings：
The just RNA that the pickup probe of structure is used in living cells is imaged.The high expression survivin and c-raf justice of selection
The palace strength cancer cell HeLa of RNA is as positive cell line, and normal breast cell MCF-10A is as negative cells system.Utilize laser
Laser Scanning Confocal Microscope carries out real time imagery to intracellular survivin justice RNA, and Cy3- ring-types are added into cell culture medium
DNA/GO is co-cultured, and HeLa observes obvious fluorescence into the cell after 15min and fluorescence intensity is as incubation time extends
And strengthen（Fig. 5 a）, and it is also very weak that the fluorescence produced after 60min is co-cultured even if addition Cy3-ssDNA/GO（Fig. 5 b）.It is worth note
Meaning, after being co-cultured with MCF-10A cells, Cy3- cyclic DNAs/GO does not observe fluorescence generation substantially, illustrates due to lacking
Weary target justice RNA is very faint in negative cells inner annular DNA/GO reasons for its use signal, in contrast, Cy3-ssDNA/
GO can produce nonspecific fluorescence signal in negative cells, this is probably due to being subject to albumen to disturb and send out in the cell
Raw enzymolysis reasons for its use fluorescence signal（Fig. 6）.Above-mentioned experiment shows, even if having using with living cells system, cyclic DNA/GO
There are the sensitivity and specificity better than ssDNA/GO.
D. Subcellular Localization：
Most foreign substance is all inevitably entered in inclusion body/lysosome after entering cell by endocytosis, so
One highly acidity and rich in enzyme microenvironment in, most of unmodified single stranded DNAs can be degraded quickly causing function to be lost.
In order to study cyclic DNA/GO into the destiny after cell, the subcellular proteomics of cyclic DNA/GO are studied, emphasis closes
The common location situation of note and inclusion body/lysosome.A period of time is being incubated altogether with Cy5- cyclic DNAs/GO or Cy5-ssDNA/GO
Afterwards, HeLa cells are dyed with LysoTracker Green dyestuffs, makes intracellular acidic organelles mark upper green glimmering
Light.Such as Fig. 7, by confocal microscopy cell it can be found that at any one time point, intracellular cyclic DNA's is glimmering
Light is all stronger than ssDNA, this is consistent with just RNA imaging results before.Importantly, by intracellular fluorescence distribution
Found after carrying out constituency analysis, cyclic DNA is very low with the fluorescence overlap proportion of inclusion body/lysosome, it is most of it was observed that
It is that red cyclic DNA fluorescence and inclusion body/lysosome fluorescence of green are individually present.Illustrate that most of cyclic DNA is to be in
Among cytoplasm, i.e. the environment that the stability of cyclic DNA/GO makes it possible to be resistant in inclusion body/lysosome is not degraded, and then
Therefrom escape into cytoplasm, and fluorescence signal is produced after being combined with target justice RNA.And the red fluorescence of ssDNA is with including
The green fluorescence of body/lysosome then occurs obvious overlapping, is due to that degraded occurs in inclusion body/lysosome to generate non-spy
The fluorescence signal of the opposite sex, the number of probes for also allowing for entering cytoplasm identification target justice RNA substantially reduce.By sub- thin
Born of the same parents distribution research, it was confirmed that cyclic DNA/GO will not be trapped in inclusion body/lysosome, make its be more suitable for it is intracellular just
Adopted RNA imagings or even gene therapy.
E. gene therapy：
In order to investigate cyclic DNA whether can as just RNA target to gene therapy medicament, we have studied cyclic DNA pair
The antisense effect of survivin and c-raf mRNA.Survivin and c-raf kinases is all during the occurrence and development of tumour
Play a very important role.Therefore, we have studied the influence that cyclic DNA/GO produces the propagation of tumour cell.With
After cyclic DNA/GO of target survivin and c-raf are incubated jointly, the measure of cell survival rate is carried out.Such as Fig. 8 a, ring-type
DNA/GO can more effectively suppress the propagation of HeLa cells than ssDNA/GO, and have concentration dependent.However, for the moon
Property cell MCF-10A, cyclic DNA/GO almost without Inhibit proliferaton effect（Fig. 9）.The above results illustrate cyclic DNA/GO energy
It is enough more effectively to suppress growth of tumour cell.Then, the gene inhibition by detected by Western blot to cyclic DNA/GO
Verified, after Fig. 8 b and 8c show that cell is incubated 24h altogether with survivin the and c-raf mRNA cyclic DNA/GO targeted,
The expression quantity of survivin and c-raf kinases is all significantly lowered, and effect is better than ssDNA/GO.To sum up, cyclic DNA/GO is to target
Gene has the inhibiting rate of higher, can result in the cell mortality of higher.
Above-mentioned analysis of experimental results shows that circular DNA probes can be used for intracellular efficient justice RNA imagings and base
Because for the treatment of.Cyclic DNA/GO has the property better than ssDNA/GO in many aspects.First, cyclic DNA/GO has the mesh of higher
Mark the recognition efficiency of justice RNA, including sensitivity, selectivity and antijamming capability.When for intracellular sensing detection, ring-type
DNA/GO can effectively eliminate false positive signal and reduce background signal.In addition, cyclic DNA/GO can be from after entering cell
Escape in inclusion body/lysosome, the suppression to just RNA translations is more efficient, therefore all has to protein expression and cell Proliferation
There is more preferable inhibition.
SEQUENCE LISTING
<110>University of Fuzhou
<120>A kind of application of cyclic DNA in the detection, imaging and gene therapy of just RNA
<130> 6
<160> 6
<170> PatentIn version 3.3
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<211> 20
<212> DNA
<213>Artificial sequence
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<212> DNA
<213>Artificial sequence
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caaggagctg caaggctggg 20
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<213>Artificial sequence
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cctgatccca gccttccagc tccttgttcc cagccttcca gctccttgtt cccagccttc 60
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<213>Artificial sequence
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cagctccttg ttagtcggta 80
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gacatgcatt ttagtcggta 80
Claims (10)
1. application of a kind of cyclic DNA in the detection or imaging of just RNA, it is characterised in that the cyclic DNA includes one
Or multiple and detection target justice RNA sequence complementation DNA probe fragment, fluorescent marker；The cyclic DNA by ssDNA from
The 3 ' of body and 5 ' end connection cyclisation are formed.
2. application of the cyclic DNA according to claim 1 in the detection or imaging of just RNA, it is characterised in that including
Following steps：（1）The cyclic DNA and fluorescence quenching are compounded to form combined probe；（2）Make the combined probe with comprising
The thing to be detected interaction of target justice RNA is detected, forms the heteroduplex of cyclic DNA and justice RNA；（3）Described in detection
The fluorescence intensity of cyclic DNA and the heteroduplex of justice RNA.
3. application of the cyclic DNA according to claim 2 in the detection or imaging of just RNA, it is characterised in that described
Fluorescence quenching has fluorescence quenching capability, and the combination of single-chain nucleic acid with double-strandednucleic acid without being combined.
4. application of the cyclic DNA according to claim 3 in the detection or imaging of just RNA, it is characterised in that described
Fluorescence quenching is graphene oxide, carbon nanotubes, azotized carbon nano piece, manganese dioxide nano-plates or boron nitride nanosheet.
5. application of the cyclic DNA according to claim 1 or 2 in the detection or imaging of just RNA, it is characterised in that
The cyclic DNA includes more than two DNA probe fragments with detection target justice RNA sequence complementation, and it is each to further include connection
The junction fragment of DNA probe fragment.
6. application of the cyclic DNA according to claim 1 in the detection or imaging of just RNA, it is characterised in that described
Just RNA is mRNA, siRNA or microRNA.
7. application of the cyclic DNA according to claim 2 in the detection or imaging of just RNA, it is characterised in that step
（2）Described in the thing to be detected comprising detection target justice RNA be cell, the combined probe is incubated jointly with cell, thin
Intracellular forms the compound of cyclic DNA and justice RNA.
8. application of a kind of cyclic DNA in the gene therapy of just RNA, it is characterised in that include the following steps：Make load institute
The nucleic acid carrier for stating cyclic DNA enters with the cell comprising detection target justice RNA, forming the miscellaneous of cyclic DNA and justice RNA
Close double-strand and depart from from the nucleic acid carrier, the just RNA in the heteroduplex is enzymatically decomposed, the expression of related gene
Journey is obstructed；The cyclic DNA includes one or more DNA probe fragments with detection target justice RNA sequence complementation；The ring
Shape DNA is formed by 3 ' and 5 ' the end connection cyclisation of ssDNA itself.
9. application of the cyclic DNA according to claim 8 in the gene therapy of just RNA, it is characterised in that the ring
Shape DNA includes more than two DNA probe fragments with detection target justice RNA sequence complementation, further includes each DNA probe of connection
The junction fragment of fragment.
10. application of the cyclic DNA according to claim 8 in the gene therapy of just RNA, it is characterised in that described
Just RNA is mRNA, siRNA or microRNA；The nucleic acid carrier for graphene oxide, carbon nanotubes, azotized carbon nano piece,
Manganese dioxide nano-plates or boron nitride nanosheet.
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| CN109295168A (en) * | 2018-10-18 | 2019-02-01 | 山东大学 | The ratio fluorescent biosensor that manganese dioxide nano-plates mediate is used for the detection and imaging of intracellular microRNA |
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| CN109295168A (en) * | 2018-10-18 | 2019-02-01 | 山东大学 | The ratio fluorescent biosensor that manganese dioxide nano-plates mediate is used for the detection and imaging of intracellular microRNA |
| CN109295168B (en) * | 2018-10-18 | 2022-02-15 | 山东大学 | Manganese dioxide nanosheet-mediated ratio fluorescence biosensor for detecting and imaging intracellular microRNA |
| CN110082531A (en) * | 2019-04-11 | 2019-08-02 | 南方医科大学南方医院 | A kind of tumour excretion body nano fluorescent detection kit and its application |
| CN110904224A (en) * | 2019-07-19 | 2020-03-24 | 中国科学院长春应用化学研究所 | Manganese dioxide/nucleic acid probe composite nano probe capable of being amplified by signal specificity and preparation method and application thereof |
| CN111234815A (en) * | 2020-01-22 | 2020-06-05 | 哈尔滨工业大学 | Preparation and use methods of biomass carbon quantum dot fluorescence detector |
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