CN117187238A - DNA shearing device and method - Google Patents
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- CN117187238A CN117187238A CN202311444019.0A CN202311444019A CN117187238A CN 117187238 A CN117187238 A CN 117187238A CN 202311444019 A CN202311444019 A CN 202311444019A CN 117187238 A CN117187238 A CN 117187238A
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- 238000000034 method Methods 0.000 title claims abstract description 37
- 230000007547 defect Effects 0.000 claims abstract description 60
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- 230000003197 catalytic effect Effects 0.000 claims abstract description 32
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 8
- 108020004414 DNA Proteins 0.000 claims description 104
- CWQXQMHSOZUFJS-UHFFFAOYSA-N molybdenum disulfide Chemical compound S=[Mo]=S CWQXQMHSOZUFJS-UHFFFAOYSA-N 0.000 claims description 57
- 229910052982 molybdenum disulfide Inorganic materials 0.000 claims description 57
- 102000053602 DNA Human genes 0.000 claims description 9
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 8
- 229910052717 sulfur Inorganic materials 0.000 claims description 8
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- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 239000010410 layer Substances 0.000 claims description 4
- 229910052750 molybdenum Inorganic materials 0.000 claims description 4
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Abstract
A DNA shearing device comprising a two-dimensional nanomaterial having a catalytically active defect therein, the catalytically active defect comprising at least one of a point defect, a line defect, and a surface defect, the two-dimensional nanomaterial shearing DNA through the catalytically active defect when in contact with the DNA. A DNA shearing method comprising the steps of: s1, preparing a two-dimensional nano material to have catalytic activity defects, wherein the catalytic activity defects comprise at least one of point defects, line defects and surface defects; s2, after the DNA solution and the two-dimensional nanomaterial are interacted, shearing DNA through the catalytic activity defect of the two-dimensional nanomaterial. The DNA shearing device and the method utilize the catalysis process of the two-dimensional nano material to shear DNA, can realize the shearing of DNA without modification of biological materials such as amino acid, RNA and the like, and have the advantages of no need of external light and metal ion drive shearing, high shearing efficiency, adjustability and the like.
Description
Technical Field
The application relates to a gene editing technology, in particular to a DNA shearing device and a method.
Background
DNA is a biological macromolecule carrying genetic information, is a material basis of the whole gene expression process, and carries information of life transmission. Restriction endonucleases can recognize specific DNA base sequences, can cleave phosphodiester bonds between two nucleotides at specific positions in each strand, making them important tools for chromosomal profiling, nucleotide sequence analysis, target gene isolation and DNA recombination. In recent years, many artificially designed biomimetic restriction endonucleases have been reported. However, their cleavage site recognition still depends on the artificial design of nucleotide sequences.
The natural enzyme has wide application due to good catalytic specificity and high catalytic efficiency, but the natural enzyme is easily inactivated by the influence of environmental conditions. Traditional mimic enzymes have catalytic properties similar to those of the natural enzymes, recognize substrates, and have enzymatic active centers. Various mimic enzymes, such as metal complexes, porphyrins, cyclodextrins, biomolecules and the like, have been developed in various fields, and have the advantages of acid resistance, alkali resistance, good thermal stability, high catalytic activity, low cost and capability of being synthesized in a large amount. If the difference of affinities between different morphologies and different crystal faces of the nano material and various DNA bases can be obtained, the morphology and crystal structure of the nano material can be selected and designed through a target shearing sequence, so that the aim of shearing any natural sequence at fixed points is fulfilled. 2018, kuang Hua et al realized that cysteine modified CdTe nanoparticles can specifically recognize and cleave at the restriction site GAT' ATC in double-stranded DNA of more than 90 base pairs after photon excitation, mimicking a restriction endonuclease, and demonstrated that photoinduced reactive oxygen species are important reasons for DNA cleavage. The DNA is sheared by the nano material, and the DNA molecule is destroyed by the active oxygen method. However, the method is harmful to organisms, and how to simulate the catalytic characteristics of natural enzymes, and the realization of the catalytic shearing of DNA by nano materials under the conditions of moderate temperature (37 ℃) and neutrality (pH= -7) is still a challenge.
It should be noted that the information disclosed in the above background section is only for understanding the background of the application and thus may include information that does not form the prior art that is already known to those of ordinary skill in the art.
Disclosure of Invention
The main object of the present application is to overcome the above-mentioned drawbacks of the background art and to provide a DNA shearing device and method.
In order to achieve the above purpose, the present application adopts the following technical scheme:
a DNA shearing device comprising a two-dimensional nanomaterial having a catalytically active defect therein, the catalytically active defect comprising at least one of a point defect, a line defect, and a surface defect, the two-dimensional nanomaterial shearing DNA through the catalytically active defect when in contact with the DNA.
Further:
the two-dimensional nano material is a molybdenum disulfide nano sheet or a single-layer molybdenum disulfide film.
The catalytic activity defect of the molybdenum disulfide nanosheets is formed by sulfur vacancies.
The edges of the molybdenum disulfide have catalytic properties that are capable of shearing DNA.
Including one, two or more layers of two-dimensional nanomaterial.
A DNA shearing method comprising the steps of:
s1, preparing a two-dimensional nano material to have catalytic activity defects, wherein the catalytic activity defects comprise at least one of point defects, line defects and surface defects;
s2, after the DNA solution and the two-dimensional nanomaterial are interacted, shearing DNA through the catalytic activity defect of the two-dimensional nanomaterial.
Further:
in the step S1, a chemical vapor deposition method is adopted to prepare a molybdenum disulfide film, and the concentration ratio of molybdenum to sulfur is 1:1-10:1.
In the step S1, the growth temperature is 810-830 ℃, preferably 820 ℃, and the cooling speed is 5-30 ℃/S, preferably 10 ℃/S.
In the step S2, the DNA solution is dripped on the molybdenum disulfide film, the incubation time is 5-30min, the temperature is 4-90 ℃, the DNA and the molybdenum disulfide are combined together, and after the interaction of the DNA and the molybdenum disulfide film, the edge catalysis of the molybdenum disulfide is utilized to shear the DNA.
The DNA is circular, linear, single-stranded or double-stranded.
The application has the following beneficial effects:
compared with the previous active oxygen DNA shearing method, the DNA shearing device and the shearing method provided by the application shear DNA by utilizing the catalysis characteristics of the inorganic two-dimensional nano material, and the method provided by the application can realize the shearing of the inorganic two-dimensional material on DNA without modification of biological materials such as amino acid, RNA and the like, and have the advantages of no need of external light and metal ion drive shearing, high shearing efficiency, adjustability and the like.
Other advantages of embodiments of the present application are further described below.
Drawings
FIG. 1 is a schematic diagram of a DNA shearing device according to an embodiment of the present application for catalyzing and shearing DNA by defects of molybdenum disulfide.
FIG. 2 is an atomic force microscope image of DNA shearing by molybdenum disulfide edge shearing of DNA in an embodiment of the present application.
FIG. 3 is a schematic diagram showing the effect of the star-shaped activity characteristic of molybdenum disulfide and the catalytic performance of molybdenum disulfide on DNA shearing by the electrophoresis technology of the DNA shearing device in the embodiment of the application.
Detailed Description
The following describes embodiments of the present application in detail. It should be emphasized that the following description is merely exemplary in nature and is in no way intended to limit the scope of the application or its applications.
It will be understood that when an element is referred to as being "mounted" or "disposed" on another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element. In addition, the connection may be for both a fixing action and a coupling or communication action.
It is to be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are merely for convenience in describing embodiments of the application and to simplify the description by referring to the figures, rather than to indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus are not to be construed as limiting the application.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the embodiments of the present application, the meaning of "plurality" is two or more, unless explicitly defined otherwise.
Referring to fig. 1 and 2, an embodiment of the present application provides a DNA shearing apparatus, including a two-dimensional nanomaterial having a catalytically active defect therein, the catalytically active defect including at least one of a point defect, a line defect, and a surface defect, and shearing DNA through the catalytically active defect when the two-dimensional nanomaterial is in contact with DNA. The DNA shearing device can shear DNA without limitation, and circular, linear, single-stranded and double-stranded DNA can be sheared.
In a preferred embodiment, the two-dimensional nanomaterial is a molybdenum disulfide nanosheet or a monolayer molybdenum disulfide film. FIG. 1 shows a schematic diagram of molybdenum disulfide shearing of DNA.
In a preferred embodiment, the catalytically active defects of the molybdenum disulfide nanoplatelets are formed from sulfur vacancies.
In a preferred embodiment, the edges of the molybdenum disulfide have catalytic properties that are capable of shearing DNA. In a preferred embodiment, the molybdenum disulfide nanoplatelets have angular edges that catalyze cleavage of DNA, as shown in fig. 2.
In various embodiments, the two-dimensional nanomaterial may include one, two, or more layers of two-dimensional nanomaterial.
The embodiment of the application also provides a DNA shearing method, which comprises the following steps:
s1, preparing a two-dimensional nano material to have catalytic activity defects, wherein the catalytic activity defects comprise at least one of point defects, line defects and surface defects;
s2, after the DNA solution and the two-dimensional nano material are interacted, for example, the DNA solution is applied to the two-dimensional nano material to enable the DNA solution and the two-dimensional nano material to interact, and DNA in the DNA solution is sheared through the catalytic activity defect of the two-dimensional nano material.
In a preferred embodiment, in step S1, a chemical vapor deposition method is used to prepare a molybdenum disulfide film, where the concentration ratio of molybdenum to sulfur is 1:1 to 10:1, and preferably 10:1.
in a preferred embodiment, in step S1, the growth temperature is 810-830 ℃, preferably 820 ℃, and the cooling rate is 5-30 ℃/S, preferably 10 ℃/S.
In addition to molybdenum disulfide nanosheets or films, other types of two-dimensional materials including heterojunction, single-atom doping, defect engineering and other materials with defect catalytic activity can be used in the DNA shearing method.
The preparation of the two-dimensional nano material can be prepared by methods such as a metal ion intercalation method, a chemical plating method, a pulse laser deposition method, a mechanical stripping method, a low-temperature ball milling method and the like besides a chemical vapor deposition method.
In a preferred embodiment, in the step S2, the DNA solution is dripped on the molybdenum disulfide film, the incubation is waited for 5-30min, the temperature is 4-90 ℃, the DNA and the molybdenum disulfide are combined together, and after the interaction of the DNA and the molybdenum disulfide film, the edge catalysis of the molybdenum disulfide is utilized to shear the DNA. FIG. 2 shows an atomic force microscope image of DNA sheared by molybdenum disulfide in accordance with an embodiment of the present application.
In other embodiments, the catalytic performance of the two-dimensional nanomaterial can be regulated and controlled by utilizing regulation and control modes such as an external light field, an electric field and the like.
In various embodiments, the DNA is circular, linear, single-stranded, or double-stranded DNA.
In a particular embodiment, the sheared DNA is plasmid pBR322.
Compared with the previous active oxygen DNA shearing method, the DNA shearing device and the shearing method provided by the application shear DNA by utilizing the catalysis characteristics of the inorganic two-dimensional nano material, and the method provided by the application can realize the shearing of the inorganic two-dimensional material on DNA without modification of biological materials such as amino acid, RNA and the like, and have the advantages of no need of external light and metal ion drive shearing, high shearing efficiency, adjustability and the like.
Specific embodiments of the present application are described further below.
In one embodiment, the DNA shearing device consists essentially of molybdenum disulfide nanoplatelets. The molybdenum disulfide film is prepared by adopting a chemical vapor deposition method, wherein the growth temperature is 820 ℃, and the concentration ratio of molybdenum to sulfur is 10:1, ensuring that more sulfur vacancies exist in the molybdenum disulfide, ensuring that the cooling speed is 10 ℃/s, ensuring better contact between the molybdenum disulfide film and the substrate, and preventing the DNA solution from washing away the molybdenum disulfide film. 1 μl of the DNA solution is dripped on the molybdenum disulfide film, waiting for 5-30min incubation time, and ensuring that the DNA and the molybdenum disulfide are combined together.
As shown in fig. 2, after the interaction of DNA with the molybdenum disulfide film, the molybdenum disulfide shears the DNA, and a distinct DNA shear boundary appears, which is caused by edge catalysis of the molybdenum disulfide.
The DNA of the examples is plasmid pBR322, and edge catalysis of molybdenum disulfide can cleave the phosphodiester bond at any position. Molybdenum disulfide transfers charge to the phosphodiester linkage of DNA, which breaks due to its instability. As shown in fig. 2, the molybdenum disulfide solution cleaves DNA near the edge where the molybdenum disulfide catalytic activity is strong by interaction with DNA. In the embodiment, as shown in fig. 3, the method of freezing and ball milling is adopted to prepare the material with more molybdenum disulfide defects, and the molybdenum disulfide solution and the DNA are mixed. As apparent from the experimental results of the examples, DNA was sheared and phosphodiester bonds were broken. Specifically, the molybdenum disulfide solution prepared by low-temperature ball milling is prepared by mixing molybdenum disulfide nanosheets with pure water, the concentration is 500mg/L, and the solution is placed for 3 days and then mixed with DNA. As shown in fig. 3, the experimental results showed that Form I bands disappeared at different incubation times of 5, 10, 20, 30 minutes, and the electrophoretogram showed asterisk activity of the molybdenum disulfide solution (non-specific site cleavage occurred) and DNA was effectively sheared.
The application utilizes molybdenum disulfide MoS 2 The active defect sites of the material realize the catalytic activity of the material, so that the DNA is sheared, and compared with the prior art, the application utilizes the catalytic activity to shear the DNA and has the advantages of no need of external illumination and the like.
The application utilizes the catalytic activity caused by the activity defect of molybdenum disulfide and MoS 2 Two-dimensional material using one, two or more layers of MoS 2 To achieve catalytic activity.
The present application provides methods of fabricating two-dimensional materials having active defects, such as using chemical vapor deposition.
Circular, linear, single-stranded, double-stranded DNA may be sheared.
The DNA shearing method of the present application does not limit the concentration of the DNA solution, and the temperature range of the solution is wide, for example, 4 to 90 ℃.
Besides molybdenum disulfide nano materials, the application can utilize other two-dimensional materials, including heterojunction, single-atom doping, defect engineering and other materials with defect catalytic activity. The application can also utilize the regulation and control modes of external light field, electric field and the like to regulate and control the catalytic performance of the nano material.
The background section of the present application may contain background information about the problems or environments of the present application and is not necessarily descriptive of the prior art. Accordingly, inclusion in the background section is not an admission of prior art by the applicant.
The foregoing is a further detailed description of the application in connection with specific/preferred embodiments, and it is not intended that the application be limited to such description. It will be apparent to those skilled in the art that several alternatives or modifications can be made to the described embodiments without departing from the spirit of the application, and these alternatives or modifications should be considered to be within the scope of the application. In the description of the present specification, reference to the terms "one embodiment," "some embodiments," "preferred embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Those skilled in the art may combine and combine the features of the different embodiments or examples described in this specification and of the different embodiments or examples without contradiction. Although embodiments of the present application and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the scope of the application as defined by the appended claims.
Claims (10)
1. A DNA shearing device comprising a two-dimensional nanomaterial having a catalytically active defect therein, the catalytically active defect comprising at least one of a point defect, a line defect, and a surface defect, the two-dimensional nanomaterial shearing DNA through the catalytically active defect when in contact with the DNA.
2. The DNA shearing device as in claim 1, wherein the two-dimensional nanomaterial is a molybdenum disulfide nanosheet or a monolayer molybdenum disulfide film.
3. The DNA shearing device as in claim 2, wherein the catalytically active defect of molybdenum disulfide is formed by a sulfur vacancy.
4. The DNA shearing device as in claim 3, wherein the edges of the molybdenum disulfide have catalytic properties capable of shearing DNA.
5. The DNA shearing device as in any of claims 1 to 4, comprising one, two or more layers of two-dimensional nanomaterial.
6. A DNA shearing method comprising the steps of:
s1, preparing a two-dimensional nano material to have catalytic activity defects, wherein the catalytic activity defects comprise at least one of point defects, line defects and surface defects;
s2, after the DNA solution and the two-dimensional nanomaterial are interacted, shearing DNA in the DNA solution through the catalytic activity defect of the two-dimensional nanomaterial.
7. The method of shearing DNA according to claim 6, wherein in step S1, a chemical vapor deposition method is used to prepare the molybdenum disulfide film, and the concentration ratio of molybdenum to sulfur is 1:1-10:1.
8. The method of shearing DNA according to claim 7, wherein in step S1, the growth temperature is 810 to 830℃and the cooling rate is 5 to 30℃per second.
9. The method according to any one of claims 7 to 8, wherein in step S2, a DNA solution is dropped onto the molybdenum disulfide film, incubating is performed for 5 to 30 minutes at a temperature of 4 to 90 ℃, the DNA and molybdenum disulfide are combined together, and after the DNA interacts with the molybdenum disulfide film, the DNA is sheared by edge catalysis of the molybdenum disulfide.
10. The DNA shearing method according to any one of claims 7 to 8, wherein the DNA is circular, linear, single-stranded or double-stranded DNA.
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