CN110079522B - Use method of monolayer molybdenum disulfide and DNA cutting method - Google Patents
Use method of monolayer molybdenum disulfide and DNA cutting method Download PDFInfo
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Abstract
The invention provides a use method of monolayer molybdenum disulfide and a DNA cutting method. The DNA cutting method provided by the invention can realize DNA cutting at normal temperature under the condition of only using a single layer of molybdenum disulfide and not adding any other auxiliary reagent.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a use method of monolayer molybdenum disulfide and a DNA cutting method.
Background
Nucleic acid is an important biological macromolecule of a living body, plays an important role in the continuation of life, the maintenance of the genetic characteristics of biological species, growth and development, cell differentiation and the like, and is also closely related to biological variation, such as tumors, genetic diseases, metabolic diseases and the like. Therefore, how to effectively manipulate nucleic acid molecules has potential application value not only in the aspect of treating diseases such as cancer, but also in biotechnology. In the early 50 s, biologists discovered restriction and modification of DNA by the behavior of phage infection. In 1970, the first restriction endonuclease Hind II was isolated from haemophilus influenzae, opening a way to cleave DNA molecules. Such enzymes recognize a particular sequence on a double-stranded DNA molecule and cleave it to form discrete fragments of a certain length and sequence. However, in recent years, the study of small organic molecules to influence and interfere with nucleic acid molecules has attracted research interest.
Among them, the research of simulating artificial nuclease of nucleic acid to effectively cut Deoxyribonucleic acid (Deoxyribonucleic acid) has achieved remarkable results; many transition metal complexes that can efficiently cleave DNA have been synthesized. For example, the classical metal complexes such as Fe (Bleomycin), mn (Porphyin) and Cu (Phenanthroline) can effectively cleave DNA. The geometric configuration of the metal complex plays an important role in the interaction process with DNA, so that the reasonable design of the metal complex not only can change the spatial configuration and the electronic structure of the metal complex, but also plays an important role in researching the action mechanism and the biological function of the metal complex and the DNA, and thus, the metal complex is used for searching multifunctional reagents capable of recognizing the conformation and the sequence of the DNA. In addition, the research on the interaction between the small molecular transition metal complex and the macromolecular DNA is used for exploring the structure, the action mechanism and the function of the macromolecular DNA, and an important theoretical basis is provided for the development and research of other research fields, such as gene chips, DNA biosensors, DNA computers and the like.
The research finds that the metal complex containing the large planar structure ligand has good capability of combining with DNA, thereby having good DNA cutting activity. However, small molecular organic compounds with super-large planar structures are synthesized, and the compounds with larger planarity are formed as ligands of metal ions, and the compounds and DNA can only react under the action of illumination or other auxiliary reagents (documents: inorg. Chem.2007,46,11122-11132, J. Phys. Chem.B 2010,114, 5851-5861. CN102286458A discloses a DNA cutting method based on metal ions and graphene oxide, which comprises the steps of preparing a graphene oxide aqueous solution, adding sterilized graphene oxide and metal ions into a DNA buffer solution, and reacting at normal temperature to realize DNA cutting; although the patent can be carried out without adding any auxiliary reagent or irradiating with light, the graphene oxide still needs to be combined with metal ions to complete the DNA cleavage.
At present, the action of the nano material such as C60, graphene oxide and the like with DNA needs to be realized under an auxiliary reagent or illumination, so that a new DNA cutting method needs to be developed.
Disclosure of Invention
The invention aims to provide a use method of monolayer molybdenum disulfide and a DNA cutting method. The DNA cutting method provided by the invention can realize DNA cutting at normal temperature under the condition of only using a single layer of molybdenum disulfide and not adding any other auxiliary reagent.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method of using a monolayer of molybdenum disulfide for cleaving DNA.
In the invention, the monolayer molybdenum disulfide can realize the cutting of DNA without adding any other auxiliary reagent. Compared with the existing small molecule DNA cutting agent, the two-dimensional nano material has a large plane and a large specific surface area, can improve the cutting effect on DNA, and further realizes the cutting of DNA.
The structure of the molybdenum disulfide is a two-dimensional nano material, and the molybdenum disulfide can induce the cutting of DNA through the generation of ROS (reactive oxygen species), so that the molybdenum disulfide can cut the DNA without adding any other auxiliary reagent.
Preferably, the using method comprises the following steps: and mixing the monolayer molybdenum disulfide with the DNA for reaction to realize the cutting of the DNA.
Preferably, the concentration of the monolayer of molybdenum disulfide in the reaction system is 1-500mg/L, such as 5mg/L, 10mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L, 250mg/L, 300mg/L, 350mg/L, 400mg/L, 450mg/L, and the like.
Within the concentration range of 1-500mg/L, the stronger the cutting effect on DNA along with the increase of the concentration, if the concentration is less than 1mg/L, the cutting of the DNA is difficult to realize because the concentration of the molybdenum disulfide is lower, and if the concentration is more than 500mg/L, the waste of the molybdenum disulfide is caused.
Preferably, the monolayer of molybdenum disulphide has a size in the range of 30-500nm, such as 40nm, 50nm, 60nm, 80nm, 100nm, 120nm, 150nm, 170nm, 200nm, 220nm, 250nm, 280nm, 300nm, 320nm, 340nm, 380nm, 400nm, 420nm, 480nm, etc.
In the range of 30-500nm, the cutting effect on DNA is enhanced with the reduction of the size of the molybdenum disulfide.
Preferably, the monolayer of molybdenum disulfide is sterilized prior to mixing with the DNA.
Preferably, the reaction system has a pH of 5 or more, for example, 6, 7, 8, 9, 10,11, 12, 13, etc., and more preferably 5 to 9.
With the increase of the pH value, the generation amount of ROS is increased, the cutting effect on DNA is gradually enhanced, and if the pH value is too small, the generation amount of ROS is too small, and the cutting effect on DNA is small.
Preferably, the reaction temperature is 10-40 ℃, such as 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ and so on.
Preferably, the reaction time is 0.5 to 30h, such as 1h, 2h, 5h, 10h, 12h, 15h, 18h, 20h, 25h, 28h, and the like.
Preferably, the DNA is added in the form of a DNA buffer.
Preferably, the buffer is PBS buffer.
Preferably, the concentration of the DNA in the reaction system is 0.01 to 0.02. Mu.g/. Mu.L, for example, 0.012. Mu.g/. Mu.L, 0.013. Mu.g/. Mu.L, 0.014. Mu.g/. Mu.L, 0.015. Mu.g/. Mu.L, 0.016. Mu.g/. Mu.L, 0.018. Mu.g/. Mu.L, or the like.
Preferably, the preparation method of the monolayer molybdenum disulfide is a lithium ion intercalation method.
The monolayer molybdenum disulfide of the present invention may be formed by any method known in the art that results in a monolayer of molybdenum disulfide, illustratively, by lithium ion intercalation.
Preferably, the DNA is selected from circular double stranded DNA.
The method for using the monolayer molybdenum disulfide provided by the invention can realize the cutting of DNA as long as the DNA has double-stranded and circular structures, and the invention can cut circular DNA into linear DNA.
As a preferred technical scheme, the using method comprises the following steps:
and mixing the monolayer molybdenum disulfide with the size of 150-350nm with the PBS buffer solution of the DNA, and reacting for 0.5-30h at the temperature of 10-40 ℃ to realize the cutting of the DNA.
Wherein the concentration of the molybdenum disulfide monolayer is 1-500mg/L, the concentration of DNA is 0.01-0.02 mu g/mu L, and the pH value of the reaction system is 5-9.
In a second aspect, the present invention provides a method for cleaving DNA, the method for cleaving DNA according to the first aspect.
Compared with the prior art, the invention has the following beneficial effects:
(1) In the invention, the monolayer molybdenum disulfide can realize the cutting of DNA without adding any other auxiliary reagent.
(2) In the invention, the concentration of the molybdenum disulfide of the single layer is within the range of 1-500mg/L, the size is within the range of 30-500nm, and the cutting effect on DNA is stronger along with the increase of the concentration and the reduction of the size of the molybdenum disulfide of the single layer; in the range of pH value of 5-9, the stronger the cutting action on DNA along with the increase of pH value; within the reaction time of 0.5-30h, the longer the reaction time, the stronger the cutting effect on DNA.
Drawings
FIG. 1 is a gel electrophoresis chart of DNAs of examples 1 and 5 to 7.
FIG. 2 is a gel electrophoresis chart of DNAs of examples 1 and 10 to 12.
FIG. 3 is a gel electrophoresis of the DNAs of examples 1 to 4.
FIG. 4 is a gel electrophoresis chart of DNAs of examples 1 and 8 to 9.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
A method for cutting DNA, which comprises the following steps:
mixing a monolayer molybdenum disulfide with the size of 225nm with a PBS buffer solution of DNA, and reacting for 18h at 25 ℃ to realize the cutting of the DNA;
wherein the concentration of the molybdenum disulfide monolayer is 50mg/L, the concentration of DNA is 0.015 mu g/mu L, the pH value of the reaction system is 7, and the DNA is pBR322.
Examples 2 to 4
The only difference from example 1 is that in this example, the size of the monolayer of molybdenum disulfide is 175nm (example 2), 300nm (example 3), 325nm (example 4).
Examples 5 to 7
The difference from example 1 is only that in this example, the concentrations of the monolayer of molybdenum disulfide were 10mg/L (example 5), 100mg/L (example 6), and 200mg/L (example 7).
Examples 8 to 9
The only difference from example 1 is that in this example, the reaction system had pH values of 5 (example 8) and 9 (example 9).
Examples 10 to 12
The only difference from example 1 is that in this example, the reaction times were 10h (example 10), 15h (example 11), 20h (example 12).
Performance testing
The performance tests were performed on the DNAs provided in the examples and on the DNAs after cleavage, as follows:
(1) Gel electrophoresis pattern: testing was performed using a zenithal energy (Tannon) gel imager;
FIG. 1 is a gel electrophoresis of DNAs of examples 1 and 5 to 7, wherein Form I indicates circular DNA, form II indicates linear DNA, lane 1 indicates pBR322 as the original DNA, lane 2 indicates the DNA cleaved in example 1, lane 3 indicates the DNA cleaved in example 5, lane 4 indicates the DNA cleaved in example 6, and lane 5 indicates the DNA cleaved in example 7, and it can be seen from FIG. 1 that the cleavage effect on DNA is gradually enhanced with the increase of the concentration of molybdenum disulfide.
FIG. 2 is a gel electrophoresis of DNAs of examples 1 and 10 to 12, wherein lane 1 is pBR322 as an original DNA, lane 2 is a DNA cleaved in example 1, lane 3 is a DNA cleaved in example 10, lane 4 is a DNA cleaved in example 11, and lane 5 is a DNA cleaved in example 12, and it can be seen from FIG. 2 that the DNA cleavage effect is gradually enhanced with the increase of the reaction time.
FIG. 3 is a gel electrophoresis of DNAs of examples 1 to 4, wherein lane 1 is original DNA pBR322, lane 2 is DNA cleaved in example 4, lane 3 is DNA cleaved in example 3, lane 4 is DNA cleaved in example 1, and lane 5 is DNA cleaved in example 2. As can be seen from FIG. 3, the cleavage effect on DNA is gradually increased as the size of molybdenum disulfide is decreased.
FIG. 4 is a gel electrophoresis diagram of DNAs of examples 1 and 8 to 9, wherein lanes 1 to 3 are pBR322 as the original DNA at pH 5, 7 and 9, lane 4 is the DNA cleaved in example 8, lane 5 is the DNA cleaved in example 1, and lane 6 is the DNA cleaved in example 9, respectively, and it can be seen from FIG. 4 that the cleavage effect on the DNA is gradually enhanced with the increase in pH.
As can be seen from FIGS. 1-4, in the present invention, molybdenum disulfide can effect the cleavage of DNA.
The applicant states that the invention is illustrated by the above examples to use the monolayer molybdenum disulfide and the DNA cleavage method of the invention, but the invention is not limited to the above process steps, which means that the invention must not be dependent on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (6)
1. A method for cutting DNA by using a monolayer of molybdenum disulfide is characterized in that: in a reaction system, mixing a single-layer molybdenum disulfide with DNA for reaction to realize the cutting of the DNA;
the preparation method of the single-layer molybdenum disulfide is a lithium ion intercalation method;
the concentration of the single-layer molybdenum disulfide is 10-500mg/L;
the size of the single-layer molybdenum disulfide is 150-350nm;
the concentration of the DNA is 0.01-0.02 mu g/mu L;
in the reaction system, the pH value is 5-9;
the reaction time is 0.5-30h;
the temperature of the reaction is 10-40 ℃.
2. The method of claim 1, wherein the monolayer of molybdenum disulfide is sterilized prior to mixing with the DNA.
3. The method of claim 1, wherein the DNA is added in the form of a DNA buffer.
4. The method of claim 3, wherein the buffer is PBS buffer.
5. The method of claim 1, wherein the DNA is selected from circular double stranded DNA.
6. The method of claim 1, wherein the method of using comprises:
mixing monolayer molybdenum disulfide with the size of 150-350nm with PBS buffer solution of DNA, and reacting at 10-40 ℃ for 0.5-30h to realize DNA cutting;
wherein the concentration of the monolayer molybdenum disulfide is 10-500mg/L, the concentration of DNA is 0.01-0.02 mu g/mu L, and the pH value of the reaction system is 5-9.
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