CN110404081A - A kind of nano-complex of DNA tetrahedron and microRNA - Google Patents
A kind of nano-complex of DNA tetrahedron and microRNA Download PDFInfo
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- CN110404081A CN110404081A CN201910794369.7A CN201910794369A CN110404081A CN 110404081 A CN110404081 A CN 110404081A CN 201910794369 A CN201910794369 A CN 201910794369A CN 110404081 A CN110404081 A CN 110404081A
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Abstract
The present invention provides the nano-complexes of a kind of DNA tetrahedron and microRNA, belong to nucleic acid molecules drug field.Nano-complex of the invention is made of microRNA and DNA tetrahedron；The DNA tetrahedron is by the single-stranded tetrahedral structure formed by base pair complementarity of 4 DNA；The microRNA be covalently attached to DNA it is tetrahedral it is single-stranded on.Nano-complex stability of the invention is strong, and it is high-efficient to enter born of the same parents, can develop into the relevant drug of various microRNA, application prospect is good.
Description
Technical field
The invention belongs to the nano combined of nucleic acid molecules drug field more particularly to a kind of DNA tetrahedron and microRNA
Object.
Background technique
MicroRNA (miRNA, miR) is the non-coding single stranded RNA that one kind is about 22nt by the length of endogenous gene
Molecule is widely present with animals and plants, has multiple biological function, participate in transcription, the post-transcriptional control of gene.
Researcher is applied to multiple fields, such as regeneration field, targeted therapy using the biological function of miRNA
Deng.But miRNA is haveed the shortcomings that and be exactly that its own stability is very poor, and it is bad to enter born of the same parents' performance as other RNA.Cause
This its multiple fields extensive use it is extremely limited.Delivery microRNA, which enters the major way taken into the cell, at present is
Liposomal delivery, cationic-liposome surface is positively charged, can with the phosphate radical of nucleic acid by electrostatic interaction, by molecule wrap up into
It is interior, liposome-nucleic acid complex is formed, can also be adsorbed by the negatively charged cell membrane in surface, then pass through fusion or cell endocytic
Effect also by osmosis, microRNA is loaded into intracellular once in a while.And the bio-toxicity of liposome is higher, therefore also not
It can be known as ideal carrier.
DNA tetrahedron (tetrahedral DNA, TDN), also known as tetrahedral framework nucleic acid (tetrahedral
Framework nucleic acids, tFNAs), tetrahedron DNA nanostructure, be it is a kind of by 4 single stranded DNAs by denaturation and
Renaturation, and then a kind of tetrahedral structure that inter-strand base complementary pairing is formed, it is readily synthesized, and biocompatibility is high, usually uses
Make the carrier of some drugs.
Have not yet to see the report that DNA tetrahedron is used as miRNA carrier.
Summary of the invention
The object of the present invention is to provide the nano-complexes of a kind of DNA tetrahedron and microRNA.
Technical solution of the present invention includes:
A kind of nano-complex, it is made of microRNA and DNA tetrahedron；
The DNA tetrahedron is by the single-stranded tetrahedral structure formed by base pair complementarity of 4 DNA；
The microRNA be covalently attached to DNA tetrahedral one it is single-stranded on.
Nano-complex as the aforementioned, the DNA single-stranded sequence such as SEQ ID NO.1-2 and SEQ ID NO.4-5 institute
Show.
Nano-complex as the aforementioned, the microRNA are covalently attached to SEQ ID NO.4.
Nano-complex as the aforementioned, the microRNA are covalently attached to 5 ' single-stranded ends of the DNA.
Nano-complex as the aforementioned, the microRNA are miR214-3p.
Nano-complex above-mentioned is preparing the purposes in anti-tumor agent.
Further, the tumour is lung cancer.
Purposes of the DNA tetrahedron in enhancing microRNA stability.
Further, the enhancing microRNA stability refers to enhancing microRNA in the environment existing for serum
Stability.
DNA tetrahedron enters the purposes in cell in promotion microRNA.
Beneficial effects of the present invention are as follows:
1) nano-complex stability of the invention is good, can be stabilized in the environment for having serum；
2) it is high-efficient to enter born of the same parents to nano-complex of the invention.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 is DNA tetrahedron and its single-stranded electrophoretogram with miRNA；The 1st swimming lane from left to right be Marker (article
Band size is 20,40,60,80,100,120,140,160,180,200,300,400,500bp respectively), the second swimming lane is S1,
Third swimming lane is S2, and third swimming lane is S3, and the 4th swimming lane is S4, and the 5th swimming lane is S3-miR214-3p, and the 6th swimming lane is TDN,
7th swimming lane is TDN-miR.
Fig. 2 is transmission electron microscope detection figure.
Fig. 3 is miRNA in 1% serum ambient concentration-time diagram
Fig. 4 is miRNA concentration-time detection figure.
Fig. 5 is electrophoretogram.
Fig. 6 is cell micro observation figure.
Fig. 7 is Flow cytometry figure.
Fig. 8 is that TDN-miR enters born of the same parents' fluorescence detection figure.
Fig. 9 is Caspase3 detection of expression figure；A, Immunofluorescence test；B, Western Blot detection；C, albumen are opposite
The quantitative statistics figure of expression quantity；D, PCR as the result is shown gene expression amount multiple variation.
Figure 10 is Bax detection of expression figure；A, Immunofluorescence test；B, Western Blot detection；C, albumen relative expression
The quantitative statistics figure of amount；D, PCR as the result is shown gene expression amount multiple variation.
Figure 11 is BCL-2 detection of expression figure；A, Immunofluorescence test；B, Western Blot detection；C, albumen is with respect to table
Up to the quantitative statistics figure of amount；D, PCR as the result is shown gene expression amount multiple variation.
TFNAs is equal with TDN in figure, and tFNAs-miR is equal with TDN-miR
Specific embodiment
The preparation method of 1 nano-complex of the present invention (TDN-miR) of embodiment
1. synthetic method
By four DNA single-stranded (S1, S2, S3-miR214-3p, S4) be dissolved in TM Buffer (10 mM Tris-HCl,
50mM MgCl2, pH=8.0) in, four DNA single-stranded final concentration of 1000nM is sufficiently mixed, and is heated rapidly to 95 DEG C of holdings
10 minutes, be cooled to 4 DEG C rapidly later and maintain 20 minutes or more, the DNA tetrahedron with miRNA can be obtained.
Four single-stranded sequences (5 ' → 3 ') are as follows:
S1:ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCC TAAGTCTGAA
(SEQ ID NO.1)；
S2:ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTCAGACTTA GGAATGTTCG
(SEQ ID NO.2)；
S3-miR214-3p:acagcaggcacagacaggcaguTTTTTACTACTATGGCGGGTG ATAAAACGTGTA
GCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC(SEQ ID NO.3)；Lower-case portion is miR214-3p sequence,
U base in miR214-3p sequence can be substituted for t base；
The sequence being mutually paired in S3-miR214-3p with S1, S2 and S4 is as follows:
S3:ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGC ATGCCCATCC
(SEQ ID NO.4)；
S4:ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCAT GCTCTTCCCG
(SEQ ID NO.5)。
2. identification
DNA tetrahedron after synthesis, visible second swimming lane of electrophoresis be S1, third swimming lane be S2, third swimming lane be S3, the 4th
Swimming lane is S4, and the 5th swimming lane is S3-miR214-3p, and the 6th swimming lane is TDN, and the 7th swimming lane is TDN-miR, passes through electrophoretogram the
The pillar location of seven swimming lanes is it can be concluded that TDN-miR is bigger compared to TDN and product is single, it is believed that miRNA has been fabricated
On to DNA tetrahedron (Fig. 1).Using transmission electron microscope observing, TDN-miR of the visible diameter between 10-20nm under mirror, smaller
Grain is TDN-miR monomer, and larger particles are TDN-miR polymer (Fig. 2).
Below by the present invention is further described in the form of experimental example.
1 serum of experimental example is incubated for experiment
1. method
The synthesis of 1.1TDN-miR
With embodiment 1.
1.2 are incubated for and detect
MiR initial concentration is 1000nM, it is mixed with serum by 99: 1 volume ratio, miR is made to be in 1% serum environment
In, it is put into 37 DEG C of incubators and is incubated for, and detected in 1min, 5min, 30min using capillary gel electrophoresis, while also will
The 1000nM miR for not being mixed with serum has carried out capillary gel electrophoresis detection.
The TDN for being 1000nM by initial concentration, miR, TDN-miR are mixed with 10% serum, are put into 37 DEG C of incubators
Middle incubation, and at 24 hours later, it is detected using agarose gel electrophoresis.
2. result
MicroRNA in the environment of 1% serum after 30 minutes i.e. have occurred it is degradable, wherein red: 1% blood not being added
Clear 1000nM miR, blue: 1% serum 1 minute 1000nM miR is added, it is green: 1% 5 minutes 1000nM of serum are added
MiR, it is yellow: 1% serum 30 minutes 1000nM miR (Fig. 3) are added.Meanwhile after being repeated a number of times Concentration Testing, make
Such as miR relative concentration-time graph of Fig. 4, it is seen that after serum is added in the short time, miR occurred immediately rapidly and
A large amount of degradation (Fig. 4).
According to Fig. 5 visible (a:TDN, b:miR, c:TDN-miR), TDN-miR and TDN are 24 small in the system of 10% serum
When after be still stabilized, and miR band is without sharp trace.TDN-miR this species complex is demonstrated greatly to improve
Stability of the microRNA in enzyme environment.
The experiment of 2 inhibiting tumour cells of experimental example
1. method
The synthesis of 1.1TDN-miR
With embodiment 1.
1.2 Inhibition test
Experimental group uses the DMEM-F12 culture solution culture lung cell A549 of the final concentration of 150nM containing TDN-miR；Control
Group handles lung cell A549 using the DMEM-F12 culture solution of the final concentration of 150nM containing TDN；Blank group, which uses, is free of TDN-
The DMEM-F12 culture solution culture lung cell A549 of miR.
After cultivating 72h, microscopically observation, and it is horizontal using Apoptosis by Flow Cytometry.
2. result
Sediments microscope inspection result is as shown in fig. 6, be far below control group and sky through the processed lung carcinoma cell quantity of TDN-miR
White group, the processed lung carcinoma cell form of TDN-miR is with respect to shrinkage in cellular morphology.
Flow cytometry results (Fig. 7) show that left lower quadrant is number of viable cells, bottom right, upper left and right upper quadrant in figure
For apoptosis and dead cell sum；Show significantly to increase through the processed lung carcinoma cell quantity apoptosis rate of TDN-miR.
Experimental example 3 enters born of the same parents' test experience
This experiment to TDN-miR enter born of the same parents carried out detect and have taken fluorescence.
1. method
Cy5 fluorescence is hung on S1 chain and synthesizes Cy5-TDN-miR, and uses the final concentration of 150nM's of Cy5-TDN-miR
DMEM-F12 culture solution culture lung cell A549, after culture 24 hours, using immunofluorescence technique to nucleus and cell bone
Frame carries out dyeing and is shot.
2. result
As a result it as shown in figure 8, red color visible fluorescence is Cy5-TDN-miR, was uniformly distributed in cytoplasm at 24 hours
Portion, it was demonstrated that it is intracellular that Cy5-TDN-miR is able to enter A549.
4 the expression of apoptosis-associated genes test experience of experimental example
1. method
Blank group is extracted, the RNA and albumen of control group and experimental group cell use quantitative fluorescent PCR and Western
Blot detects apoptosis-related genes caspase-3 respectively, and Bax, Bcl-2 express (transcription and translation level).
2. result
Such as Fig. 9 B, shown in 10B, the addition of TDN-miR promotes the apoptosis of cell.Increase the egg of caspase3 and Bax
White expression intensity.In Fig. 9 D, 10D, the result of q-pcr also shows that identical trend, and Figure 11 B presses down apoptogene Bcl-2
Protein expression intensity decline, in Figure 11 D, the result of q-pcr also shows that identical trend.Western blot result and
It analyzes column Fig. 9 C, 10C, 11C, and as a result same immunofluorescence results (Fig. 9 A, 10A, 11A) are consistent.Data are expressed as average value
± SD (n=4).Student t is examined for statisticalling analyze.Statistical analysis: * p < 0.05, * * p < 0.01, * * * p < 0.001.
To sum up, stability of the microRNA in serum can be greatly enhanced in DNA tetrahedron of the invention, and promotes
MicroRNA enters cells play effect, has a good application prospect.
SEQUENCE LISTING
<110>Sichuan University
<120>a kind of nano-complex of DNA tetrahedron and microRNA
<130> GYKH1118-2019P017776CC
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 63
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 1
atttatcacc cgccatagta gacgtatcac caggcagttg agacgaacat tcctaagtct 60
gaa 63
<210> 2
<211> 63
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 2
acatgcgagg gtccaatacc gacgattaca gcttgctaca cgattcagac ttaggaatgt 60
tcg 63
<210> 3
<211> 90
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 3
acagcaggca cagacaggca gttttttact actatggcgg gtgataaaac gtgtagcaag 60
ctgtaatcga cgggaagagc atgcccatcc 90
<210> 4
<211> 63
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 4
actactatgg cgggtgataa aacgtgtagc aagctgtaat cgacgggaag agcatgccca 60
tcc 63
<210> 5
<211> 63
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 5
acggtattgg accctcgcat gactcaactg cctggtgata cgaggatggg catgctcttc 60
ccg 63
Claims (10)
1. a kind of nano-complex, which is characterized in that it is made of microRNA and DNA tetrahedron；
The DNA tetrahedron is by the single-stranded tetrahedral structure formed by base pair complementarity of 4 DNA；
The microRNA be covalently attached to DNA tetrahedral one it is single-stranded on.
2. nano-complex as described in claim 1, which is characterized in that the DNA single-stranded sequence such as SEQ ID NO.1-2
With shown in SEQ ID NO.4-5.
3. nano-complex as claimed in claim 2, which is characterized in that the microRNA is covalently attached to SEQ ID
NO.4。
4. the nano-complex as described in claim 1 is any, which is characterized in that the microRNA is covalently attached to described
5 ' DNA single-stranded ends.
5. the nano-complex as described in claim 1-4, which is characterized in that the microRNA is miR214-3p.
6. any nano-complex of claim 1-5 is preparing the purposes in anti-tumor agent.
7. purposes as claimed in claim 6, which is characterized in that the tumour is lung cancer.
Purposes of the 8.DNA tetrahedron in enhancing microRNA stability.
9. purposes as claimed in claim 8, which is characterized in that the enhancing microRNA stability refers to enhancing
Stability of the microRNA in the environment existing for serum.
10.DNA tetrahedron enters the purposes in cell in promotion microRNA.
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Cited By (5)
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| CN113736776A (en) * | 2021-09-03 | 2021-12-03 | 四川大学 | MicroRNA nano complex based on framework nucleic acid material and preparation method and application thereof |
| CN114748604A (en) * | 2022-05-10 | 2022-07-15 | 四川大学 | Compound for bone marrow damage and/or inhibition |
| CN115068496A (en) * | 2022-06-29 | 2022-09-20 | 四川大学 | Compound of DNA tetrahedral framework nucleic acid and typhaneoside and application thereof in preparing medicine for treating acute kidney injury |
| CN116139285A (en) * | 2022-07-07 | 2023-05-23 | 四川大学 | Biological switchable miR nano-composite based on DNA tetrahedral framework nucleic acid and preparation method and application thereof |
| CN117695301A (en) * | 2023-12-08 | 2024-03-15 | 哈尔滨医科大学附属第一医院 | Framework nucleic acid-based miRNA-21 delivery system and application thereof in repairing corneal epithelial damage |
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| CN114748604A (en) * | 2022-05-10 | 2022-07-15 | 四川大学 | Compound for bone marrow damage and/or inhibition |
| CN114748604B (en) * | 2022-05-10 | 2023-04-07 | 四川大学 | Compound for bone marrow damage and/or inhibition |
| CN115068496A (en) * | 2022-06-29 | 2022-09-20 | 四川大学 | Compound of DNA tetrahedral framework nucleic acid and typhaneoside and application thereof in preparing medicine for treating acute kidney injury |
| CN115068496B (en) * | 2022-06-29 | 2023-07-07 | 四川大学 | Complex of DNA tetrahedral framework nucleic acid and typha neoglycoside and application thereof in preparing medicine for treating acute kidney injury |
| CN116139285A (en) * | 2022-07-07 | 2023-05-23 | 四川大学 | Biological switchable miR nano-composite based on DNA tetrahedral framework nucleic acid and preparation method and application thereof |
| CN116139285B (en) * | 2022-07-07 | 2024-05-31 | 四川大学 | Biological switchable miR nano-composite based on DNA tetrahedral framework nucleic acid and preparation method and application thereof |
| CN117695301A (en) * | 2023-12-08 | 2024-03-15 | 哈尔滨医科大学附属第一医院 | Framework nucleic acid-based miRNA-21 delivery system and application thereof in repairing corneal epithelial damage |
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