Application of a low-dimensional nanomaterial in DNA specific site recognition and cleavage

Abstract

The present invention discloses an application of a low-dimensional nanomaterial in recognition and shearing of DNA specific sites, wherein a low-dimensional nanomaterial solution is mixed with a DNA solution, thereby utilizing the catalytic activity of the low-dimensional nanomaterial to recognize and shear the specific sites of DNA; wherein the DNA has at least one specific site consisting of at least 7 consecutive bases T. The present invention has the advantages of not requiring external light and metal ions to drive shearing, not requiring modification of organic materials such as amino acids, high shearing efficiency, high temperature tolerance, etc., and realizes recognition and shearing of DNA specific sites by inorganic low-dimensional nanomaterials.

Description

Technical Field

[0001] The present invention relates to the field of gene editing, and in particular to the application of a low-dimensional nanomaterial in the recognition and shearing of DNA specific sites. Background Art

[0002] DNA is a biological macromolecule that carries genetic information. It is the material basis of the entire gene expression process and carries the information transmitted by life. Restriction endonucleases can recognize specific DNA base sequences and cut the phosphodiester bond between two nucleotides at specific locations in each chain, making them important tools for chromosome map analysis, 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 relies on artificially designed nucleotide sequences.

[0003] Natural enzymes are widely used due to their good catalytic specificity and high catalytic efficiency, but they are easily inactivated by environmental conditions. Traditional enzyme mimics have catalytic properties similar to natural enzymes, can recognize substrates, and have enzyme catalytic activity centers. People have developed various enzyme mimics in various fields, such as metal complexes, porphyrins, cyclodextrins, biomolecules, etc., which are acid-resistant, alkali-resistant, thermally stable, have high catalytic activity, low cost, and can be synthesized in large quantities. If the differences in affinity between different morphologies and different crystal faces of nanomaterials and various DNA bases can be obtained, the morphology and crystal structure of nanomaterials can be selected and designed through the target shearing sequence, so as to achieve the purpose of point-by-point shearing of any natural sequence. In 2018, Kuang Hua et al. realized that cysteine-modified CdTe nanoparticles can specifically recognize and cut at the restriction site GAT′ATC in double-stranded DNA of more than 90 base pairs after photon excitation, mimicking restriction endonucleases, and proved that light-induced reactive oxygen is an important reason for cutting DNA. However, there is still a lack of research on the use of nanomaterials for site-specific cleavage of natural DNA sequences.

[0004] It should be noted that the information disclosed in the above background technology section is only used for understanding the background of the present application, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the present invention provides an application of a low-dimensional nanomaterial in the recognition and shearing of DNA specific sites.

[0006] The present invention adopts the following technical solution:

[0007] A low-dimensional nanomaterial is used for the recognition and shearing of DNA specific sites. A low-dimensional nanomaterial solution is mixed with a DNA solution, thereby utilizing the catalytic activity of the low-dimensional nanomaterial to recognize and shear the specific sites of the DNA; wherein the DNA has a specific site consisting of at least 7 consecutive bases T.

[0008] Preferably, the low-dimensional nanomaterial is at least one of a two-dimensional nanomaterial, a one-dimensional nanomaterial, and a zero-dimensional nanomaterial.

[0009] Preferably, the low-dimensional nanomaterial has at least one of point defects, line defects and surface defects.

[0010] Preferably, the concentration of the low-dimensional nanomaterial solution is 200 mg / L-1000 mg / L; the concentration of the DNA solution is 300 ng / μL-700 ng / μL.

[0011] Preferably, the low-dimensional nanomaterial is molybdenum disulfide nanosheets.

[0012] Preferably, the DNA is a pBR322 plasmid.

[0013] Preferably, the solvents in the low-dimensional nanomaterial solution and the DNA solution are both pure water.

[0014] Preferably, the low-dimensional nanomaterial solution and the DNA solution are mixed for 5 min-3 h at a temperature of 4-85°C.

[0015] Preferably, the molybdenum disulfide nanosheets are prepared by low-temperature ball milling; preferably, the conditions of the low-temperature ball milling are: filled with liquid nitrogen, milled 15 times per minute, and the total grinding time is 150-300 minutes.

[0016] Preferably, the thickness of the molybdenum disulfide nanosheets is between 0.6 nm and 20 nm.

[0017] The present invention has a beneficial effect: the present invention mixes a low-dimensional nano material solution with a DNA solution, thereby utilizing the catalytic activity of the low-dimensional nano material to identify and shear DNA-specific sites (composed of at least 7 consecutive bases T). Specifically, the DNA is sheared by utilizing the catalytic process when the low-dimensional nano material acts on the DNA. It has been experimentally verified that the efficiency of the embodiment of the present invention in shearing DNA is 20%-50% within the range of 5-30 minutes, and the temperature tolerance is high. Therefore, compared with the existing DNA shearing technology, the present invention has the advantages of not requiring external light and metal ions to drive shearing, not requiring modification of organic materials such as amino acids, high shearing efficiency, high temperature tolerance, etc., and realizes the recognition and shearing of DNA-specific sites by inorganic low-dimensional nano materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the process of recognition and shearing of DNA specific sites by molybdenum disulfide nanosheets in a preferred embodiment of the present invention.

[0019] Figure 2 This is a thickness distribution diagram of molybdenum disulfide nanosheets measured by atomic particle microscope in a preferred embodiment of the present invention.

[0020] Figure 3 The electrophoretic diagrams of Examples 1-4 of the present invention at different incubation times are shown.

[0021] Figure 4 This is a percentage diagram corresponding to the Fragments of Examples 1-4 of the present invention.

[0022] Figure 5 The electrophoresis diagrams of Examples 5-8 of the present invention at different temperatures are shown. DETAILED DESCRIPTION

[0023] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope and application of the present invention. In the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other.

[0024] A specific embodiment of the present invention provides an application of a low-dimensional nanomaterial in the recognition and shearing of DNA specific sites, wherein a low-dimensional nanomaterial solution is mixed with a DNA solution, thereby utilizing the catalytic activity of the low-dimensional nanomaterial to recognize and shear the specific sites of the DNA; wherein the DNA has a specific site composed of at least 7 consecutive bases T (i.e., at least 7 TTTTTTT).

[0025] In a preferred embodiment, the low-dimensional nanomaterial is at least one of a two-dimensional nanomaterial (such as a planar material, a heterojunction, a single atom-doped two-dimensional nanomaterial, etc.), a one-dimensional nanomaterial (such as a tubular or linear material, a single atom-doped one-dimensional nanomaterial), and a zero-dimensional nanomaterial (such as a point material, a single atom-doped zero-dimensional nanomaterial, etc.).

[0026] In a preferred embodiment, the low-dimensional nanomaterial has at least one of point defects, line defects and surface defects.

[0027] In a preferred embodiment, the low-dimensional nanomaterial can be prepared by chemical vapor deposition, lithium ion intercalation, chemical electroplating, pulsed laser deposition, mechanical exfoliation, low-temperature ball milling, and the like.

[0028] In a preferred embodiment, the concentration of the low-dimensional nanomaterial solution is 200 mg / L-1000 mg / L, for example, the concentration can be 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, 700 mg / L, 800 mg / L, 900 mg / L, 1000 mg / L, etc.; the concentration of the DNA solution is 300 ng / μL-700 ng / μL, for example, the concentration can be 300 ng / μL, 400 ng / μL, 500 ng / μL, 600 ng / μL, 700 ng / μL, etc.

[0029] In a preferred embodiment, the low-dimensional nanomaterial is molybdenum disulfide nanosheets.

[0030] In a preferred embodiment, the DNA is a pBR322 plasmid.

[0031] In a preferred embodiment, the solvents in the low-dimensional nanomaterial solution and the DNA solution are both pure water.

[0032] In a preferred embodiment, the low-dimensional nanomaterial solution and the DNA solution are mixed for 5 min-3 h at a temperature of 4-85°C.

[0033] In a preferred embodiment, the molybdenum disulfide nanosheets are prepared by low-temperature ball milling; preferably, the conditions of the low-temperature ball milling are: filled with liquid nitrogen, grinding 15 times per minute, and the total grinding time is 150-300 minutes.

[0034] In a preferred embodiment, the thickness of the molybdenum disulfide nanosheets is between 0.6 nm and 20 nm.

[0035] The following uses MoS2 nanosheets (two-dimensional materials) as an example to illustrate its application in the recognition and shearing of DNA-specific sites. The schematic diagram of the recognition and shearing process of MoS2 nanosheets on DNA-specific sites is as follows: Figure 1 As shown, the molybdenum disulfide nanosheets are mainly composed of sulfur vacancies (a combination of point defects and line defects), and the catalytic process when it interacts with DNA recognizes and cuts the DNA specific site TTTTTTT. Specifically, it includes the following steps:

[0036] 1. Prepare MoS2 nanosheets by low temperature ball milling (put 10g MoS2 powder into a ball mill, fill it with liquid nitrogen, grind 15 times per minute, grind for 150min in total), ensuring that MoS2 has strong edge activity and a thickness in the range of 1-20nm, such as Figure 2 Shown is the thickness distribution of MoS2 nanosheets measured by atomic force microscopy.

[0037] 2. Use ultrapure water to prepare a molybdenum disulfide nanosheet solution with a concentration of 100-1000 mg / L.

[0038] 3. Mix the molybdenum disulfide nanosheet solution and DNA (DNA is pBR322 plasmid) solution (solvent is ultrapure water) and incubate at 4-85°C for 5min-3h, then load nucleic acid dye for electrophoresis analysis. Figure 3 The following is a specific experimental example:

[0039] Experimental Example 1: A molybdenum disulfide nanosheet solution (molybdenum disulfide concentration of 200 mg / L) and a pBR322 plasmid solution (concentration of 500 ng / μl) were mixed and incubated at room temperature (20-25° C.) for 5 min.

[0040] Experimental Example 2: The difference from Experimental Example 1 is that the incubation time is 10 minutes.

[0041] Experimental Example 3: The difference from Experimental Example 1 is that the incubation time is 20 minutes.

[0042] Experimental Example 4: The difference from Experimental Example 1 is that the incubation time is 30 minutes.

[0043] like Figure 3 As shown, it is the electrophoresis diagram of Examples 1-4 at different incubation times, wherein Form1 represents supercoiled DNA (i.e., pBR322 plasmid in the example), Fragment is the sheared DNA fragment, ladder is the standard ruler, and lane is the electrophoresis channel (channels 1-5 represent Form1 and Examples 1-4, respectively, which have not been treated by the embodiments of the present invention). Figure 4 As shown, it is the percentage corresponding to the Fragment in Examples 1-4. Figure 3 and 4 It can be seen that by comparing lanes 2-5 and lane 1, after DNA is mixed with molybdenum disulfide, molybdenum disulfide shears DNA, and a new band fragment appears. Further combined with the Sanger sequencing technology, the two ends of the fragment are 7 Ts, as shown in Table 1 below. By comparing the fragment and the ladder, it can be seen that the length of the fragment is about 1000bp. The percentage of the fragment in Lane 2-5 can be calculated based on the gray value, thereby determining that the shearing efficiency of Examples 1-4 is within the range of 30%-50%, as shown in Table 1. Figure 4 shown.

[0044] Table 1: Sequencing results of DNA fragments cut from pBR322 plasmid

[0045]

[0046]

[0047] Note: In the DNA fragment (5'-3') in Table 1, the TTTTTTT at both ends are not included in the fragment. As can be seen from Table 1, in the 5'-3' chain of the DNA sequence of the pBR322 plasmid, the two TTTTTTTs are cleavage sites. Through the action of the molybdenum disulfide nanosheets and the pBR322 plasmid, the DNA fragment between the two TTTTTTTs can be cut off to obtain the fragment of the sequence shown in Table 1 except for the TTTTTTT at both ends.

[0048] Experimental Example 5: A solution of molybdenum disulfide nanosheets (molybdenum disulfide concentration of 200 mg / L) and a solution of pBR322 plasmid (concentration of 500 ng / μl) were mixed and incubated at 22° C. for 5 min.

[0049] Experimental Example 6: The difference from Experimental Example 5 is that the incubation temperature is 40°C.

[0050] Experimental Example 7: The difference from Experimental Example 5 is that the incubation temperature is 70°C.

[0051] Experimental Example 8: The difference from Experimental Example 5 is that the incubation temperature is 85°C.

[0052] like Figure 5 As shown, the electrophoresis diagrams of Examples 5-8 at different temperatures, channels 1-5 represent Form 1 and Examples 5-8, respectively, which have not been treated by the embodiments of the present invention. Figure 5 It can be seen that as the temperature increases, the catalytic activity of the molybdenum disulfide material is not lost, and it exhibits star activity. However, natural nucleases are inactivated at high temperatures. Therefore, in the application of the embodiments of the present invention, molybdenum disulfide has a higher tolerance to temperature.

[0053] The specific embodiment of the present invention utilizes DNA and MoS2 specific physical adsorption to identify the TTTTTTT site, and utilizes the catalytic activity of molybdenum disulfide to shear DNA. Compared with the prior art, it has the advantages of specific site recognition, DNA catalytic shearing, and high shearing efficiency.

[0054] In other embodiments, other low-dimensional nanomaterials with defects can be used to replace the molybdenum disulfide nanosheets to perform DNA site-specific shearing.

[0055] The above content is a further detailed description of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, without departing from the concept of the present invention, it can also make several substitutions or modifications to these described embodiments, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention. In the description of this specification, the description of the reference terms "an embodiment", "some embodiments", "preferred embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily target the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In the absence of mutual contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. An application of a low-dimensional nanomaterial in the recognition and shearing of DNA specific sites, characterized in that: The low-dimensional nanomaterial solution is mixed with the DNA solution, so as to utilize the catalytic activity of the low-dimensional nanomaterial to recognize and cut the specific site TTTTTTT of the DNA; The low-dimensional nanomaterial is a molybdenum disulfide nanosheet, which is prepared by a low-temperature ball milling method. The conditions of the low-temperature ball milling are: filled with liquid nitrogen, milled 15 times per minute, and the total milling time is 150-300 minutes to ensure that the molybdenum disulfide has strong edge activity and a thickness within the range of 1-20nm. The DNA is a pBR322 plasmid.

2. The use of the low-dimensional nanomaterial as claimed in claim 1 in recognition and shearing of DNA specific sites, characterized in that: The concentration of the low-dimensional nano material solution is 200 mg / L-1000 mg / L; the concentration of the DNA solution is 300 ng / μL-700 ng / μL.

3. The use of the low-dimensional nanomaterial as claimed in claim 1 in recognition and shearing of DNA specific sites, characterized in that: The solvents in the low-dimensional nanomaterial solution and the DNA solution are both pure water.

4. The use of the low-dimensional nanomaterial as claimed in claim 1 in recognition and shearing of DNA specific sites, characterized in that: The time for mixing the low-dimensional nano material solution and the DNA solution is 5 minutes to 3 hours, and the temperature is 4-85°C.

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