CN108186666A - The application of a kind of DNA nanobelts on anti-apoptotic and preparation method thereof - Google Patents
The application of a kind of DNA nanobelts on anti-apoptotic and preparation method thereof Download PDFInfo
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- CN108186666A CN108186666A CN201810083270.1A CN201810083270A CN108186666A CN 108186666 A CN108186666 A CN 108186666A CN 201810083270 A CN201810083270 A CN 201810083270A CN 108186666 A CN108186666 A CN 108186666A
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- 230000002424 anti-apoptotic effect Effects 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
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- 239000000243 solution Substances 0.000 claims description 18
- 239000011777 magnesium Substances 0.000 claims description 13
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- 238000006243 chemical reaction Methods 0.000 claims description 6
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 6
- 229940069446 magnesium acetate Drugs 0.000 claims description 6
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- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 14
- 238000005516 engineering process Methods 0.000 abstract description 7
- 108060006006 Cytochrome-c peroxidase Proteins 0.000 abstract description 4
- 238000012377 drug delivery Methods 0.000 abstract description 4
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract description 2
- 230000008685 targeting Effects 0.000 abstract description 2
- 230000006907 apoptotic process Effects 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 10
- 102100030497 Cytochrome c Human genes 0.000 description 6
- 108010075031 Cytochromes c Proteins 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
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- 238000010521 absorption reaction Methods 0.000 description 3
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- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
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- 108090000397 Caspase 3 Proteins 0.000 description 2
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- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 2
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
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- Health & Medical Sciences (AREA)
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- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
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Abstract
The invention belongs to biomedicine technical field, the DNA nanobelts that a kind of DNA paper foldings technology synthesizes are applied in anti-apoptotic by the application of more particularly to a kind of DNA nanobelts on anti-apoptotic and preparation method thereof.The present invention inhibits cytochrome C peroxidase activity using DNA origami structures, so as to achieve the purpose that anti-apoptotic, is expected to provide important references in terms of the drug delivery system and targeting drug delivery regulated and controled for cromoci enzymatic activity.
Description
Technical field
The invention belongs to biomedicine technical field, application of more particularly to a kind of DNA nanobelts on anti-apoptotic
And preparation method thereof.
Background technology
Apoptosis refers to for human internal environment is maintained to stablize, and is cell by the death of the apoptotic of gene control
A kind of basic biological phenomena, but disorderly apoptosis process has with many diseases and directly or indirectly closes
System.Wherein, cromoci is to participate in the indispensable important factor of Apoptosis, and cromoci is embedding on mitochondrial inner membrane
Enter albumen, on the outside of mitochondrial inner membrane, there is heme group, as a single electron carrier in membrane-bound complex
Electronics is transmitted between III (Cyt c reductases) and compound IV (Cyt c oxidizing ferment).It can cause line grain when apoptotic signal causes
Cytochrome c release in body, cytochrome c and the apoptosis enzyme activition factor etc. form apoptosis body, then active cell apoptosis mistake
The most important shearing of end eventually enzyme caspase-3 in journey, and then cause caspases cascade reactions, lead to Apoptosis.At present, press down
The several method of Apoptosis processed has：(1) cytochrome c is eliminated from cytosol by immunodepletion；(2) in cytosol
Middle addition sucrose inhibits cytochrome c to be released to stablize mitochondria；(3) using apoptosis inhibitory protein (AP families), in cell
Inhibit caspase-3 on the spindle of m period, the activity of caspase-7 continues down mitosis；(4)
- 2 gene of bone-marrow-derived lymphocyte knurl discharged from cell mitochondrial using inhibition cromoci is played usually in the form of dimer
(Bcl-2).Above several conventional methods are comparatively laborious, and bad control, some destination proteins are difficult to prepare and are difficult to put into city
Field is promoted, and effect is uncontrollable.For example, the expressing gene of the survivin albumen in AP families is only expressed in tumour and embryo's group
It knits, is expressed seldom in normal adult tissue, although the albumen can be made under existing technical conditions, but needed in body
The complicated genetic transcription of outer simulation and expression environment, are a bigger challenges so that the albumen is difficult to be made；Bcl-2 this
The mostly existing Anti-G value of the substance of one family also plays the role of promoting apoptosis, and control is improper to be likely to result in reverse effect.
DNA paper folding technologies are exactly DNA pictures using the specific binding between DNA molecular structure in itself and its molecule
" paper " is equally converted into various 2D and 3D structures, which causes DNA not just for a kind of inhereditary material, more to have developed DNA more
For complicated structure and function, under conditions of DNA paper folding technologies are gradually improved, the structure and function of DNA is more and more diversified.
Research shows that when apoptotic signal causes, in mitochondria, permeability conversion hole (PTP) between outer membrane is open, carefully
Cytochrome C is discharged, and combined with the apoptotic proteins hydrolase activation factor in endochylema by transposition from mitochondria, and then is started
Caspase cascade reactions, promote Apoptosis.
The present invention by a kind of existing DNA origami structures apply with the improper apoptotic process of cell, by inhibiting cell color
Plain C peroxidase activities are come caspase cascade reactions that cromoci is inhibited to be started, so as to reach anti-apoptotic
Purpose.
Invention content
The present invention inhibits cytochrome C peroxidase activity using DNA origami structures, so as to reach anti-apoptotic
Purpose is expected to provide important references in terms of the drug delivery system and targeting drug delivery regulated and controled for cromoci enzymatic activity.
The present invention provides a kind of application of DNA nanobelts on anti-apoptotic, and the DNA nanobelts be by
SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, four DNA chain described in SEQ ID NO.4 are prepared.
The preparation method of DNA nanobelts, includes the following steps in one such application：
S1 prepares following four DNA chain that concentration is 100 μm of ol/L, spare：
Staple 1:5 '-cagccctgtaagatgaagatagcgtctatgcc-3 ',
Staple 2:5 '-ccctgactcaca atggtcggattccgtctctg-3 ',
Staple 3:5 '-tctcaacttcaactcgtattctca actcgtat-3 ',
Scaffold strand:
5’-cagggctgggcatagaagtcagggcagagacgagttgagaatacgagttgagaatacgagttgaga
atccgaccattgtgcgctatcttcatctta-3’；
10 × TA/Mg solution is prepared by formula as below：2.42g tris, 1.34g tetra-s' water magnesium acetate, 40ml ultra-pure waters are used
Acetic acid adjusts and is settled to 50ml with ultra-pure water after its pH is 7.4；
Four DNA chain in S1 are respectively taken 1 μ L to add in the 10 × TA/Mg solution prepared in the S1 of 10 μ L, then added in by S2
86 μ L ultra-pure waters, obtain mixed liquor；
S3, the mixed liquor in S2 is reacted, and reaction condition is：85 DEG C -20 DEG C are declined with 1 DEG C of gradient, Mei Gewen
It spends gradient and keeps 2min, 4 DEG C of preservations obtain DNA nanobelts.
Compared with prior art, beneficial effects of the present invention：
What DNA origami structures were started by inhibiting cytochrome C peroxidase activity to inhibit cromoci
Caspase cascade reactions so as to achieve the purpose that anti-apoptotic, provide a kind of convenient and easy, stability and high efficiency, accurate controllable
Inhibition Apoptosis drug.
Description of the drawings
Fig. 1 is the ultraviolet absorptivity curve graph that DNR of the present invention inhibits cytochrome C peroxidase activity；
Fig. 2 is the circular dichroism figure that DNR of the present invention influences Cyt C protein structure；
Fig. 3 is the structure diagram of DNR of the present invention.
Specific embodiment
Several specific embodiments of the present invention are described in detail in 1-3 below in conjunction with the accompanying drawings, it is to be understood that this hair
Bright protection domain is not restricted by specific implementation, and the reagent involved in embodiment can be obtained by public channel.
Embodiment 1
A kind of application of DNA nanobelts (hereinafter referred to as DNR) on anti-apoptotic, and DNA nanobelts are by SEQ
ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 four DNA chain be prepared.The DNA nanobelts
It is to be synthesized by DNA paper folding technologies.
Above-mentioned DNR preparation processes are as follows：
S1 prepares following four DNA chain (artificial synthesized by Invitrogen companies) that concentration is 100 μm of ol/L,
It is spare：
Staple 1:5 '-cagccctgtaagatgaagatagcgtctatgcc-3 ', as shown in SEQ ID NO.1；
Staple 2:5 '-ccctgactcaca atggtcggattccgtctctg-3 ', as shown in SEQ ID NO.2；
Staple 3:5 '-tctcaacttcaactcgtattctca actcgtat-3 ', as shown in SEQ ID NO.3；
Scaffold strand:
5’-cagggctgggcatagaagtcagggcagagacgagttgagaatacgagttgagaatacgagttgaga
Atccgacc attgtgcgctatcttcatctta-3 ', as shown in SEQ ID NO.4；
10 × TA/Mg solution is prepared by formula as below：2.42g tris, 1.34g tetra-s' water magnesium acetate, 40ml ultra-pure waters are used
Acetic acid adjusts and is settled to 50ml with ultra-pure water after its pH is 7.4；
Four DNA chain in S1 are respectively taken 1 μ L to add in the 10 × TA/Mg solution prepared in the S1 of 10 μ L, then added in by S2
86 μ L ultra-pure waters, obtain mixed liquor；
S3, the mixed liquor in S2 is reacted, and reaction condition is：85 DEG C -20 DEG C are declined with 1 DEG C of gradient, Mei Gewen
It spends gradient and keeps 2min, 4 DEG C of preservations obtain DNA nanobelts, which can utilize PCR instrument to complete.
It should be noted that：Such as the planar structure schematic diagram that Fig. 3 is DNR of the present invention, DNR of the present invention is parallel spiral by three
The repetition rectangular unit composition of structural domain, DNA spiral of each structural domain containing one section of 48bp.In each rectangular unit,
The DNA chain of 96 base length is as stent chain (scaffold strand).In three short dna chains containing 32 bases
(staple1staple2staple3) it with the help of, is carried out with raster filling pattern.Adjacent unit is connected by three staples
It is connected together to form a long strip.
For the application effect of the verification present invention, dependence test has been done using ultraviolet dynamic method：
(1) the guaiacol solution of 20mmol/L is prepared.
(2) hydrogenperoxide steam generator of 50mmol/L is prepared.
(3) 10 × TA/Mg solution is prepared：Tetra- water magnesium acetate of 2.42g tris, 1.34g is added in, adds in 40ml ultra-pure waters, is used
Acetic acid adjusts after its pH is 7.4 and is settled to 50ml.
(4) cytochrome c solution of a concentration of 400 μm of ol/L of 17.5 μ L, a concentration of 2 μ of 17.5 μ L are added in centrifuge tube
The DNR of mol/L, 10 × TA/Mg that 25 μ L steps (3) are prepared, 190 μ L ultra-pure waters, total volume are 250 μ L, and one is incubated at 4 DEG C
Night must be incubated DNR solution.
(5) cytochrome c solution of a concentration of 400 μm of ol/L of 17.5 μ L, 25 μ L steps are added in another centrifuge tube
(3) 10 × TA/Mg prepared, 207.5 μ L ultra-pure waters, total volume are 250 μ L, and a night is incubated at 4 DEG C, obtains incubated cell pigment
C protein solution.
(6) hydrogenperoxide steam generator of 50 μ L steps (2) preparation is added in two ultraviolet microcolorimetric wares, then wherein
The incubation DNR solution of step (4) is added in one ultraviolet microcolorimetric ware, step is added in another ultraviolet microcolorimetric ware
(5) incubated cell pigment C protein solution, then the guaiacol solution of 50 μ L steps (1) preparation is added respectively, with ultraviolet power
Method monitors absorbance change respectively under the Single wavelength of 470nm.As a result such as Fig. 1.
The slope size of ultraviolet absorption curve has reacted the size of enzyme's reaction speeding, as can be seen from Figure 1 cell color
Be not added in plain C the curve of DNR under the Detection wavelength of 470nm and guaiacol, hydrogen peroxide enzymatic reaction during purple
Outer absorption and rate are more than the UV absorption and rate of cromoci after addition DNR in cromoci, this illustrates the present invention's
DNR structures have apparent inhibiting effect for the peroxidase activity of cromoci.
Embodiment 2
A kind of application of DNA nanobelts (being known as DNR) on anti-apoptotic, and the DNA nanobelts are by SEQ
ID NO.1, SEQ ID NO.2, SEQ ID NO.3, four DNA chain described in SEQ ID NO.4 are prepared.The DNA receives
Rice band is synthesized by DNA paper folding technologies.
Above-mentioned DNR nanobelts preparation process is as follows：
S1 prepares following four DNA chain (artificial synthesized by Invitrogen companies) that concentration is 100 μm of ol/L,
It is spare：
Staple 1:5 '-cagccctgtaagatgaagatagcgtctatgcc-3 ', as shown in SEQ ID NO.1；
Staple 2:5 '-ccctgactcaca atggtcggattccgtctctg-3 ', as shown in SEQ ID NO.2；
Staple 3:5 '-tctcaacttcaactcgtattctca actcgtat-3 ', as shown in SEQ ID NO.3；
Scaffold strand:
5’-cagggctgggcatagaagtcagggcagagacgagttgagaatacgagttgagaatacgagttgaga
Atccgacc attgtgcgctatcttcatctta-3 ', as shown in SEQ ID NO.4；
10 × TA/Mg solution is prepared by formula as below：2.42g tris, 1.34g tetra-s' water magnesium acetate, 40ml ultra-pure waters are used
Acetic acid adjusts and is settled to 50ml with ultra-pure water after its pH is 7.4；
Four DNA chain in S1 are respectively taken 1 μ L to add in the 10 × TA/Mg solution prepared in the S1 of 10 μ L, then added in by S2
86 μ L ultra-pure waters, obtain mixed liquor；
S3, the mixed liquor in S2 is reacted, and reaction condition is：85 DEG C -20 DEG C are declined with 1 DEG C of gradient, Mei Gewen
It spends gradient and keeps 2min, 4 DEG C of preservations obtain DNA nanobelts, which can utilize PCR instrument to complete.
For the application effect of the verification present invention, dependence test has been done using circular dichroism spectra method：
(1) 1 × TA/Mg solution is prepared：0.242g tris, 0.134g tetra-s' water magnesium acetate, 40ml ultra-pure waters, with acetic acid tune
It saves after its pH is 7.4 and is settled to 50ml.
(2) cromoci of 4 μm of ol/L is prepared with 1 × TA/Mg solution that step (1) is prepared
Solution 350 μ L are incubated a night at 4 DEG C, obtain sample 1.
(3) 350 μ L, final concentration of 0.02 μm of ol/L DNR and 4 μm of ol/L cromocis are prepared
Mixed liquor, be incubated a night at 4 DEG C, obtain sample 2.
(4) sample 1, sample 2 are monitored respectively with circular dichroism instrument, setting wavelength is 190nm-250nm, as a result as schemed
2。
As can be seen from Figure 2 for the result that circular dichroism is reflected, the cell in the sample 2 after adding in DNR
There is no change for the two level protein structure of pigment C, it was demonstrated that and the DNR designed by us does not destroy the structure of biological body protein,
Biological cell activity will not be damaged, illustrates that DNR is without side-effects to organism.
To sum up, DNR can effectively reduce peroxidase activity, and without side-effects, be in terms of being applied to biological medicine
It is very advantageous.
It should be noted that the step method used in claims of the present invention is same as the previously described embodiments, in order to anti-
Only repeat, description of the invention preferred embodiment, but those skilled in the art once know it is basic creative general
It reads, then other change and modification can be made to these embodiments.So appended claims are intended to be construed to include preferably in fact
It applies example and falls into all change and modification of the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
God and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Sequence table
<120>The application of a kind of DNA nanobelts on anti-apoptotic and preparation method thereof
<141> 2018-01-19
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213>Artificial sequence
<400> 1
cagccctgta agatgaagat agcgtctatg cc 32
<210> 2
<211> 32
<212> DNA
<213>Artificial sequence
<400> 2
ccctgactca caatggtcgg attccgtctc tg 32
<210> 3
<211> 32
<212> DNA
<213>Artificial sequence
<400> 3
tctcaacttc aactcgtatt ctcaactcgt at 32
<210> 4
<211> 96
<212> DNA
<213>Artificial sequence
<400> 4
cagggctggg catagaagtc agggcagaga cgagttgaga atacgagttg agaatacgag 60
ttgagaatcc gaccattgtg cgctatcttc atctta 96
Claims (2)
1. a kind of application of DNA nanobelts on anti-apoptotic, and the DNA nanobelts are by SEQ ID NO.1, SEQ
ID NO.2, SEQ ID NO.3, four DNA chain described in SEQ ID NO.4 are prepared.
2. application as described in claim 1, which is characterized in that the preparation method of the DNA nanobelts includes the following steps：
S1 prepares following four DNA chain that concentration is 100 μm of ol/L, spare：
Staple 1:5 '-cagccctgtaagatgaagatagcgtctatgcc-3 ',
Staple 2:5 '-ccctgactcaca atggtcggattccgtctctg-3 ',
Staple 3:5 '-tctcaacttcaactcgtattctca actcgtat-3 ',
Scaffold strand:
5’-cagggctgggcatagaagtcagggcagagacgagttgagaatacgagttgagaatacgagttgagaatcc
gaccattgtgcgctatcttcatctta-3’；
10 × TA/Mg solution is prepared by formula as below：2.42g tris, 1.34g tetra-s' water magnesium acetate, 40ml ultra-pure waters, use acetic acid
It adjusts and is settled to 50ml with ultra-pure water after its pH is 7.4；
Four DNA chain in S1 are respectively taken 1 μ L to add in the 10 × TA/Mg solution prepared in the S1 of 10 μ L, then add in 86 μ L by S2
Ultra-pure water obtains mixed liquor；
S3, the mixed liquor in S2 is reacted, and reaction condition is：85 DEG C -20 DEG C are declined with 1 DEG C of gradient, each temperature ladder
Degree keeps 2min, and 4 DEG C of preservations obtain DNA nanobelts.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810083270.1A CN108186666B (en) | 2018-01-29 | 2018-01-29 | Application of DNA nanobelt in resisting apoptosis and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810083270.1A CN108186666B (en) | 2018-01-29 | 2018-01-29 | Application of DNA nanobelt in resisting apoptosis and preparation method thereof |
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| Publication Number | Publication Date |
|---|---|
| CN108186666A true CN108186666A (en) | 2018-06-22 |
| CN108186666B CN108186666B (en) | 2020-02-21 |
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ID=62590927
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| CN201810083270.1A Expired - Fee Related CN108186666B (en) | 2018-01-29 | 2018-01-29 | Application of DNA nanobelt in resisting apoptosis and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110640158A (en) * | 2019-09-24 | 2020-01-03 | 西北大学 | Copper nanocluster synthesized by DNA nanobelt template method, and synthesis method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1703428A (en) * | 2002-10-09 | 2005-11-30 | 比奥西斯特玛股份公司 | Peptides modulating caspase activation |
| CN101791304A (en) * | 2009-08-27 | 2010-08-04 | 中山大学 | Application of Xyloketal B in preparing medicines for treating mitochondrial injury diseases |
| CN105004703A (en) * | 2015-06-26 | 2015-10-28 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for simulation of DNA nano origami structure as drug carrier by DAPI embedding and release |
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Patent Citations (3)
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|---|---|---|---|---|
| CN1703428A (en) * | 2002-10-09 | 2005-11-30 | 比奥西斯特玛股份公司 | Peptides modulating caspase activation |
| CN101791304A (en) * | 2009-08-27 | 2010-08-04 | 中山大学 | Application of Xyloketal B in preparing medicines for treating mitochondrial injury diseases |
| CN105004703A (en) * | 2015-06-26 | 2015-10-28 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for simulation of DNA nano origami structure as drug carrier by DAPI embedding and release |
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| QUAN CHEN, ET AL.: "Redox Regulation of Apoptosis before and after Cytochrome C Release", 《KOREAN JOURNAL OF BIOLOGICAL SCIENCES》 * |
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|---|---|---|---|---|
| CN110640158A (en) * | 2019-09-24 | 2020-01-03 | 西北大学 | Copper nanocluster synthesized by DNA nanobelt template method, and synthesis method and application thereof |
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