CN108186666B - Application of DNA nanobelt in resisting apoptosis and preparation method thereof - Google Patents
Application of DNA nanobelt in resisting apoptosis and preparation method thereof Download PDFInfo
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- 230000000694 effects Effects 0.000 claims abstract description 19
- 108060006006 Cytochrome-c peroxidase Proteins 0.000 claims abstract description 8
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- 239000012498 ultrapure water Substances 0.000 claims description 16
- 239000011777 magnesium Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 11
- 239000011259 mixed solution Substances 0.000 claims description 8
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- 229940097364 magnesium acetate tetrahydrate Drugs 0.000 claims description 6
- XKPKPGCRSHFTKM-UHFFFAOYSA-L magnesium;diacetate;tetrahydrate Chemical compound O.O.O.O.[Mg+2].CC([O-])=O.CC([O-])=O XKPKPGCRSHFTKM-UHFFFAOYSA-L 0.000 claims description 6
- 238000012377 drug delivery Methods 0.000 abstract description 4
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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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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to an application of a DNA nanobelt in resisting apoptosis and a preparation method thereof. The invention utilizes the DNA origami structure to inhibit the activity of cytochrome C peroxidase so as to achieve the aim of resisting apoptosis, and is expected to provide important reference in the aspects of drug delivery systems and targeted drug delivery aiming at the regulation and control of the activity of cytochrome C peroxidase.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of a DNA nanobelt in resisting apoptosis and a preparation method thereof.
Background
Apoptosis is a basic biological phenomenon of cells, and is genetically controlled to maintain homeostasis, but a disordered apoptosis process is directly or indirectly related to the occurrence of many diseases. Among them, cytochrome C is an essential factor involved in apoptosis, and cytochrome C is an insertion protein on the inner membrane of mitochondria, is located outside the inner membrane of mitochondria, has a heme group, and serves as a single electron carrier to transfer electrons between membrane-bound complex iii (Cyt C reductase) and complex iv (Cyt C oxidase). When the apoptosis signal is initiated, cytochrome c in mitochondria is released, the cytochrome c and an apoptosis enzyme activating factor and the like form an apoptosis body, then the final shear enzyme caspase-3 which is the most main in the apoptosis process is activated, and the cascade reaction of caspases is further initiated to cause the apoptosis of the cells. Currently, several methods of inhibiting apoptosis are: (1) elimination of cytochrome c from the cytosol by immunodepletion; (2) adding sucrose to the cytosol to stabilize mitochondria and inhibit cytochrome c release; (3) using apoptosis inhibiting protein (AP family) to inhibit the activity of caspase-3 and caspase-7 on the spindle body of cell in mitosis period to make mitosis proceed; (4) the B-lymphoma-2 gene (Bcl-2), which normally functions as a dimer to inhibit the release of cytochrome C from the cell mitochondria, is used. The traditional methods are complicated and difficult to control, some target proteins are difficult to prepare and difficult to put into market for popularization, and the effect is uncontrollable. For example, the expression gene of survivin protein in the AP family is only expressed in tumor and embryonic tissues, but is rarely expressed in normal adult tissues, and although the protein can be prepared under the existing technical conditions, it is a relatively large challenge to simulate a complex gene transcription and expression environment in vitro, making the protein difficult to prepare; bcl-2 is a family of substances which have both anti-apoptotic and pro-apoptotic effects, and improper control may cause adverse effects.
The DNA paper folding technology is to fold DNA into various 2D and 3D structures like paper by utilizing the specific combination between the structure of DNA molecules and the molecules, so that the DNA is not only a genetic material, but also more complex structures and functions of the DNA are developed, and the structures and functions of the DNA are more and more diversified under the condition that the DNA paper folding technology is improved.
Research shows that when an apoptosis signal is initiated, Permeability Transition Pores (PTP) between outer membranes in mitochondria are opened, cytochrome C is released from mitochondria through translocation and is combined with an apoptosis proteolytic enzyme activating factor in cytoplasm, and caspase cascade reaction is further started to promote apoptosis.
The invention applies the existing DNA paper folding structure and the abnormal apoptosis process of cells, and inhibits the caspase cascade reaction started by cytochrome C by inhibiting the activity of cytochrome C peroxidase, thereby achieving the purpose of resisting apoptosis.
Disclosure of Invention
The invention utilizes the DNA origami structure to inhibit the activity of cytochrome C peroxidase so as to achieve the aim of resisting apoptosis, and is expected to provide important reference in the aspects of drug delivery systems and targeted drug delivery aiming at the regulation and control of the activity of cytochrome C peroxidase.
The invention provides an application of a DNA nano-belt in resisting apoptosis, and the DNA nano-belt is prepared by four DNA chains described by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
A preparation method of the DNA nanobelt in the application comprises the following steps:
s1, preparing the following four DNA strands with the concentration of 100 mu mol/L for later use:
Staple 1:5’-cagccctgtaagatgaagatagcgtctatgcc-3’，
Staple 2:5’-ccctgactcaca atggtcggattccgtctctg-3’，
Staple 3:5’-tctcaacttcaactcgtattctca actcgtat-3’，
Scaffold strand:
5’-cagggctgggcatagaagtcagggcagagacgagttgagaatacgagttgagaatacgagttgagaatccgacc attgtgcgctatcttcatctta-3’；
a10 XTA/Mg solution is prepared according to the following formula: 2.42g tris, 1.34g magnesium acetate tetrahydrate and 40ml ultrapure water, adjusting the pH value to 7.4 by using acetic acid, and then fixing the volume to 50ml by using the ultrapure water;
s2, adding 1 mu L of each of the four DNA chains in the S1 into 10 mu L of 10 XTA/Mg solution prepared in the S1, and then adding 86 mu L of ultrapure water to obtain a mixed solution;
s3, reacting the mixed solution in the S2 under the following reaction conditions: the temperature of 85-20 ℃ is reduced by 1 ℃ gradient, each temperature gradient is kept for 2min, and the DNA nanobelt is obtained after storage at 4 ℃.
Compared with the prior art, the invention has the beneficial effects that:
the DNA paper folding structure inhibits caspase cascade reaction started by cytochrome C by inhibiting the activity of cytochrome C peroxidase, thereby achieving the aim of resisting apoptosis and providing a convenient, easy, stable, efficient, accurate and controllable medicine for inhibiting apoptosis.
Drawings
FIG. 1 is a graph of UV absorbance of DNR of the present invention for inhibiting cytochrome C peroxidase activity;
FIG. 2 is a circular dichroism spectrum of the effect of DNR of the present invention on cytochrome C protein structure;
FIG. 3 is a schematic structural diagram of DNR of the present invention.
Detailed Description
Several embodiments of the present invention will be described in detail below with reference to fig. 1-3, but it should be understood that the scope of the present invention is not limited to the embodiments, and the reagents involved in the examples can be obtained through common channels.
Example 1
The application of a DNA nano-belt (hereinafter referred to as DNR) in resisting apoptosis is prepared from four DNA chains of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4. The DNA nanobelt is synthesized by a DNA paper folding technology.
The DNR preparation procedure described above was as follows:
s1, four DNA strands (artificially synthesized by Invitrogen corporation) were prepared at 100. mu. mol/L for use:
staple 1: 5'-cagccctgtaagatgaagatagcgtctatgcc-3', shown in SEQ ID NO. 1;
2, 5'-ccctgactcaca atggtcggattccgtctctg-3' as shown in SEQ ID NO. 2;
staple 3: 5'-tctcaacttcaactcgtattctca actcgtat-3', shown in SEQ ID NO. 3;
Scaffold strand:
5'-cagggctgggcatagaagtcagggcagagacgagttgagaatacgagttgagaatacgagttgagaatccgacc attgtgcgctatcttcatctta-3', as shown in SEQ ID NO. 4;
a10 XTA/Mg solution is prepared according to the following formula: 2.42g tris, 1.34g magnesium acetate tetrahydrate and 40ml ultrapure water, adjusting the pH value to 7.4 by using acetic acid, and then fixing the volume to 50ml by using the ultrapure water;
s2, adding 1 mu L of each of the four DNA chains in the S1 into 10 mu L of 10 XTA/Mg solution prepared in the S1, and then adding 86 mu L of ultrapure water to obtain a mixed solution;
s3, reacting the mixed solution in the S2 under the following reaction conditions: the temperature of 85-20 ℃ is reduced by 1 ℃ gradient, each temperature gradient is kept for 2min, and the DNA nanobelt is obtained after storage at 4 ℃, and the reaction can be completed by utilizing a PCR instrument.
It should be noted that: FIG. 3 is a schematic plan view of the structure of the DNR of the present invention, which consists of three repeating rectangular units of parallel helical domains, each containing a 48bp DNA helix. In each rectangular unit, a 96-base long DNA strand serves as a scaffold strand (scaffold strand). This was done in a raster-fill mode with the aid of three 32-base short DNA strands (staple1staple2staple 3). Adjacent units are connected together by three staples to form a long strip.
In order to verify the application effect of the invention, relevant tests are carried out by applying an ultraviolet kinetic method:
(1) prepare 20mmol/L guaiacol solution.
(2) A50 mmol/L hydrogen peroxide solution was prepared.
(3) Preparing a 10 XTA/Mg solution: 2.42g of tris, 1.34g of magnesium acetate tetrahydrate and 40ml of ultrapure water were added, and the pH was adjusted to 7.4 with acetic acid to make a volume of 50 ml.
(4) To the tube was added 17.5. mu.L of a cytochrome C solution having a concentration of 400. mu. mol/L, 17.5. mu.L of DNR having a concentration of 2. mu. mol/L, 25. mu.L of 10 XTA/Mg prepared in step (3), and 190. mu.L of ultrapure water in a total volume of 250. mu.L, and the mixture was incubated at 4 ℃ overnight to obtain an incubated DNR solution.
(5) To another centrifuge tube, 17.5. mu.L of a cytochrome C solution having a concentration of 400. mu. mol/L, 25. mu.L of 10 XTA/Mg prepared in step (3) and 207.5. mu.L of ultrapure water were added in a total volume of 250. mu.L, and incubated at 4 ℃ overnight to obtain an incubated cytochrome C protein solution.
(6) And (3) adding 50 mu L of hydrogen peroxide solution prepared in the step (2) into each of the two ultraviolet micro cuvettes, then adding the incubation DNR solution in the step (4) into one of the ultraviolet micro cuvettes, adding the incubation cytochrome C protein solution in the step (5) into the other ultraviolet micro cuvette, then respectively adding 50 mu L of guaiacol solution prepared in the step (1), and respectively monitoring the absorbance change under the single wavelength of 470nm by using an ultraviolet kinetic method. The results are shown in FIG. 1.
The slope of the ultraviolet absorption curve reflects the rate of the enzymatic reaction, and it can be seen from fig. 1 that the ultraviolet absorption and rate of the curve without adding DNR in cytochrome C at the detection wavelength of 470nm and during the enzymatic reaction of guaiacol and hydrogen peroxide are greater than those of cytochrome C after adding DNR in cytochrome C, which shows that the DNR structure of the present invention has obvious inhibition effect on the peroxidase activity of cytochrome C.
Example 2
The application of the DNA nanobelt (named DNR) in resisting apoptosis is prepared from four DNA chains shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4. The DNA nanobelt is synthesized by a DNA paper folding technology.
The preparation steps of the DNR nanobelt are as follows:
s1, four DNA strands (artificially synthesized by Invitrogen corporation) were prepared at 100. mu. mol/L for use:
staple 1: 5'-cagccctgtaagatgaagatagcgtctatgcc-3', shown in SEQ ID NO. 1;
2, 5'-ccctgactcaca atggtcggattccgtctctg-3' as shown in SEQ ID NO. 2;
staple 3: 5'-tctcaacttcaactcgtattctca actcgtat-3', shown in SEQ ID NO. 3;
Scaffold strand:
5'-cagggctgggcatagaagtcagggcagagacgagttgagaatacgagttgagaatacgagttgagaatccgacc attgtgcgctatcttcatctta-3', as shown in SEQ ID NO. 4;
a10 XTA/Mg solution is prepared according to the following formula: 2.42g tris, 1.34g magnesium acetate tetrahydrate and 40ml ultrapure water, adjusting the pH value to 7.4 by using acetic acid, and then fixing the volume to 50ml by using the ultrapure water;
s2, adding 1 mu L of each of the four DNA chains in the S1 into 10 mu L of 10 XTA/Mg solution prepared in the S1, and then adding 86 mu L of ultrapure water to obtain a mixed solution;
s3, reacting the mixed solution in the S2 under the following reaction conditions: the temperature of 85-20 ℃ is reduced by 1 ℃ gradient, each temperature gradient is kept for 2min, and the DNA nanobelt is obtained after storage at 4 ℃, and the reaction can be completed by utilizing a PCR instrument.
In order to verify the application effect of the invention, a circular dichroism method is applied to make relevant tests:
(1) preparing a 1 XTA/Mg solution: 0.242g of tris, 0.134g of magnesium acetate tetrahydrate and 40ml of ultrapure water were adjusted to pH 7.4 with acetic acid and then the volume was adjusted to 50 ml.
(2) Preparing 4 mu mol/L cytochrome C by using the 1 XTA/Mg solution prepared in the step (1)
350 μ L of the solution was incubated overnight at 4 ℃ to obtain sample 1.
(3) 350 μ L of DNR at a final concentration of 0.02 μmol/L and 4 μmol/L of cytochrome C were prepared
The mixture was incubated overnight at 4 ℃ to obtain sample 2.
(4) The sample 1 and the sample 2 were monitored by a circular dichroism spectrometer, respectively, and the wavelength was set to 190nm to 250nm, and the results are shown in FIG. 2.
From the results reflected in the circular dichroism spectrum, it can be seen in fig. 2 that the secondary protein structure of cytochrome C in sample 2 after DNR addition was not changed, demonstrating that the DNR designed by us did not damage the structure of the biogenic protein and does not impair the cellular activity of the organism, indicating that DNR has no side effect on the organism.
In conclusion, DNR can effectively reduce peroxidase activity without side effects, and is very advantageous for application in biomedicine.
It should be noted that the steps and methods adopted in the claims of the present invention are the same as those of the above-mentioned embodiments, and for the sake of avoiding redundancy, the present invention describes the preferred embodiments, but those skilled in the art can make other changes and modifications to these embodiments once they learn the basic inventive concept. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<120> application of DNA nanobelt in anti-apoptosis and preparation method thereof
<141>2018-01-19
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cagccctgta agatgaagat agcgtctatg cc 32
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ccctgactca caatggtcgg attccgtctc tg 32
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tctcaacttc aactcgtatt ctcaactcgt at 32
<210>4
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cagggctggg catagaagtc agggcagaga cgagttgaga atacgagttg agaatacgag 60
ttgagaatcc gaccattgtg cgctatcttc atctta 96
Claims (2)
1. An application of a DNA nano-belt in preparing a preparation for inhibiting the activity of cytochrome C peroxidase is disclosed, and the DNA nano-belt is prepared by four DNA chains of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
2. The use of claim 1, wherein the DNA nanobelt is prepared by a method comprising the steps of:
s1, preparing the following four artificially synthesized DNA chains with the concentration of 100 mu mol/L for later use:
Staple 1:5’-cagccctgtaagatgaagatagcgtctatgcc-3’，
Staple 2:5’-ccctgactcaca atggtcggattccgtctctg-3’，
Staple 3:5’-tctcaacttcaactcgtattctca actcgtat-3’，
Scaffold strand:
5’-cagggctgggcatagaagtcagggcagagacgagttgagaatacgagttgagaatacgagttgagaatccgaccattgtgcgctatcttcatctta-3’；
a10 XTA/Mg solution is prepared according to the following formula: 2.42g tris, 1.34g magnesium acetate tetrahydrate and 40ml ultrapure water, adjusting the pH value to 7.4 by using acetic acid, and then fixing the volume to 50ml by using the ultrapure water;
s2, adding 1 mu L of each of the four DNA chains in the S1 into 10 mu L of 10 XTA/Mg solution prepared in the S1, and then adding 86 mu L of ultrapure water to obtain a mixed solution;
s3, reacting the mixed solution in the S2 under the following reaction conditions: the temperature of 85-20 ℃ is reduced by 1 ℃ gradient, each temperature gradient is kept for 2min, and the DNA nanobelt is obtained after storage at 4 ℃.
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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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Patent 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 |
Non-Patent Citations (1)
| Title |
|---|
| Redox Regulation of Apoptosis before and after Cytochrome C Release;Quan Chen, et al.;《Korean Journal of Biological Sciences》;20031231;第7卷(第1期);第1-9页 * |
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