Use of low-dimensional nanomaterial in recognition and cleavage of specific DNA locus

Abstract

Disclosed in the present invention is a use of a low-dimensional nanomaterial in recognition and cleavage of a specific DNA locus. A low-dimensional nanomaterial solution is mixed with a DNA solution, so as to recognize and cleave a specific DNA locus by using the catalytic activity of the low-dimensional nanomaterial, wherein the DNA has at least one specific locus consisting of at least 7 consecutive T bases. The present invention has the advantages of no need of external light and metal ions to drive cleavage, no need of modification of organic materials such as amino acid, high cleavage efficiency, high temperature tolerance and the like, and achieves recognition and cleavage of the specific DNA locus by using the inorganic low-dimensional nanomaterial.

Description

Application of a low-dimensional nanomaterial in DNA site-specific recognition and cleavage 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 and serves as the material foundation for the entire gene expression process, carrying the information transmitted by life. Restriction endonucleases can recognize specific DNA base sequences and cleave the phosphodiester bond between two nucleotides at specific locations within each strand, making them important tools for chromosome mapping, 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 excellent catalytic specificity and high catalytic efficiency. However, natural enzymes are easily inactivated by environmental conditions. Traditional enzyme mimics exhibit catalytic properties similar to natural enzymes, capable of substrate recognition and possessing enzymatic active sites. A variety of enzyme mimics have been developed in various fields, such as metal complexes, porphyrins, cyclodextrins, and biomolecules. These mimics exhibit acid and alkali resistance, excellent thermal stability, high catalytic activity, low cost, and can be synthesized in large quantities. If the differences in affinity between different morphologies and crystal faces of nanomaterials for various DNA bases can be understood, the morphology and crystal structure of the nanomaterials can be selected and designed based on the target cleavage sequence, thereby achieving site-specific cleavage of any natural sequence. In 2018, Kuang Hua et al. demonstrated that cysteine-modified CdTe nanoparticles can specifically recognize and, upon photon excitation, cleave at the restriction site GAT′ATC in double-stranded DNA exceeding 90 base pairs, mimicking restriction endonucleases. They also demonstrated that light-induced reactive oxygen species are a key factor in DNA cleavage. However, there is still a lack of research on the use of nanomaterials for site-directed cleavage of natural DNA sequences.

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

[0005] Summary of the Invention

[0006] 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.

[0007] The present invention adopts the following technical solutions:

[0008] A low-dimensional nanomaterial is used for the recognition and cleavage 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 cleave the specific sites of the DNA; wherein the DNA has a specific site consisting of at least 7 consecutive bases T.

[0009] Preferably, the low-dimensional nanomaterial is at least one of a two-dimensional nanomaterial, a one-dimensional nanomaterial, and a zero-dimensional nanomaterial.

[0010] Preferably, the low-dimensional nanomaterial has at least one of point defects, line defects and surface defects.

[0011] 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.

[0012] Preferably, the low-dimensional nanomaterial is molybdenum disulfide nanosheets.

[0013] Preferably, the DNA is pBR322 plasmid.

[0014] Preferably, the solvent in the low-dimensional nanomaterial solution and the DNA solution is pure water.

[0015] Preferably, the low-dimensional nanomaterial solution and the DNA solution are mixed for 5 min-3 h at a temperature of 4-85°C.

[0016] Preferably, the molybdenum disulfide nanosheets are prepared by low-temperature ball milling; preferably, the low-temperature ball milling conditions are: filled with liquid nitrogen, milling 15 times per minute, and a total milling time of 150-300 minutes.

[0017] Preferably, the thickness of the molybdenum disulfide nanosheets is between 0.6 nm and 20 nm.

[0018] The present invention has beneficial effects: the present invention mixes a low-dimensional nanomaterial solution with a DNA solution, thereby utilizing the catalytic activity of the low-dimensional nanomaterial 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 nanomaterial acts on the DNA. Experimental verification shows 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 to existing DNA shearing technologies, 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 nanomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG1 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.

[0020] FIG2 is a thickness distribution diagram of MoS2 nanosheets measured by atomic particle microscopy in a preferred embodiment of the present invention.

[0021] FIG3 is an electrophoresis diagram of Examples 1-4 of the present invention at different incubation times.

[0022] FIG4 is a percentage diagram corresponding to the fragments of Examples 1-4 of the present invention.

[0023] FIG5 is an electrophoresis diagram of Examples 5-8 of the present invention at different temperatures. DETAILED DESCRIPTION

[0024] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is merely illustrative 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 this application can be combined with each other.

[0025] A specific embodiment of the present invention provides an application of a low-dimensional nanomaterial in the recognition and cleavage 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 cleave 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).

[0026] 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.).

[0027] In a preferred embodiment, the low-dimensional nanomaterial has at least one of point defects, line defects and surface defects.

[0028] In a preferred embodiment, the low-dimensional nanomaterials can be prepared by chemical vapor deposition, lithium ion intercalation, chemical electroplating, pulsed laser deposition, mechanical exfoliation, low-temperature ball milling, and the like.

[0029] 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.

[0030] In a preferred embodiment, the low-dimensional nanomaterial is molybdenum disulfide nanosheets.

[0031] In a preferred embodiment, the DNA is a pBR322 plasmid.

[0032] In a preferred embodiment, the solvent in the low-dimensional nanomaterial solution and the DNA solution is pure water.

[0033] 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.

[0034] In a preferred embodiment, the molybdenum disulfide nanosheets are prepared by low-temperature ball milling; preferably, the low-temperature ball milling conditions are: filled with liquid nitrogen, milling 15 times per minute, and a total milling time of 150-300 minutes.

[0035] In a preferred embodiment, the thickness of the molybdenum disulfide nanosheets is between 0.6 nm and 20 nm.

[0036] The following uses molybdenum disulfide nanosheets (two-dimensional materials) as an example to illustrate their application in recognizing and shearing DNA-specific sites. A schematic diagram of the process of MoS2 nanosheets recognizing and shearing DNA-specific sites is shown in Figure 1. MoS2 nanosheets are mainly composed of sulfur vacancies (a combination of point defects and line defects). The catalytic process when interacting with DNA recognizes and shears the DNA-specific site TTTTTTT. The specific steps include the following:

[0037] 1. MoS2 nanosheets were prepared by cryogenic ball milling (10 g of MoS2 powder was placed in a ball mill filled with liquid nitrogen and milled 15 times per minute for 150 min). This ensured that the MoS2 had strong edge activity and a thickness in the range of 1-20 nm. Figure 2 shows the thickness distribution of the MoS2 nanosheets measured by atomic particle microscopy.

[0038] 2. Prepare a molybdenum disulfide nanosheet solution with ultrapure water at a concentration of 100-1000 mg / L.

[0039] 3. Mix the MoS2 nanosheet solution and DNA (DNA is pBR322 plasmid) solution (solvent is ultrapure water) and incubate at 4-85°C for 5 min-3 h, then load with nucleic acid dye for electrophoresis analysis. As shown in Figure 3, the following is a specific experimental example:

[0040] 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 minutes.

[0041] Experimental Example 2: The difference from Experimental Example 1 is that the incubation time is 10 minutes.

[0042] Experimental Example 3: The difference from Experimental Example 1 is that the incubation time is 20 minutes.

[0043] Experimental Example 4: The difference from Experimental Example 1 is that the incubation time is 30 minutes.

[0044] As shown in Figure 3, it is an electrophoresis diagram of Examples 1-4 at different incubation times, where Form 1 represents supercoiled DNA (i.e., the pBR322 plasmid in the example), Fragment is the sheared DNA fragment, Ladder is a standard ruler, and Lane is an electrophoresis channel (Channels 1-5 represent Form 1 and Examples 1-4, respectively, which have not been processed by the embodiments of the present invention). As shown in Figure 4, it is the percentage corresponding to the Fragment in Examples 1-4. As can be seen from Figures 3 and 4, by comparing lanes 2-5 and lane 1, it was found that after the DNA was mixed with molybdenum disulfide, molybdenum disulfide sheared the DNA, and a new band Fragment appeared. Further combined with Sanger sequencing technology, the two endpoints of the Fragment were 7 Ts, as shown in Table 1 below. By comparing the Fragment and Ladder, it can be seen that the length of the Fragment is approximately 1000bp. According to the grayscale value, the percentage of the fragment in Lane 2-5 can be calculated, thereby determining that the shearing efficiency of Examples 1-4 is within the range of 30%-50%, as shown in Figure 4.

[0045] Table 1: Sequencing results of DNA fragments cut from pBR322 plasmid

[0046] Note: In the DNA fragments (5'-3') in Table 1, the two TTTTTTT residues are not included in the fragment. As shown in Table 1, the two TTTTTTT residues in the 5'-3' strand of the pBR322 plasmid DNA sequence serve as cleavage sites. The interaction of the MoS2 nanosheets with the pBR322 plasmid excised the DNA fragment between the two TTTTTTT residues, resulting in the fragments shown in Table 1, excluding the TTTTTTT residues at both ends.

[0047] Experimental Example 5: 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 22° C. for 5 min.

[0048] Experimental Example 6: The difference from Experimental Example 5 is that the incubation temperature is 40°C.

[0049] Experimental Example 7: The difference from Experimental Example 5 is that the incubation temperature is 70°C.

[0050] Experimental Example 8: The difference from Experimental Example 5 is that the incubation temperature is 85°C.

[0051] Figure 5 shows electrophoretic patterns of Examples 5-8 at different temperatures, with lanes 1-5 representing Form 1 and Examples 5-8, respectively, which were not treated with the present invention. As shown in Figure 5, the catalytic activity of the molybdenum disulfide material remains unchanged with increasing temperature, exhibiting star activity. However, natural nucleases are inactivated at high temperatures. Therefore, in the applications of the present invention, molybdenum disulfide exhibits a higher temperature tolerance.

[0052] 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 cleave DNA. Compared with the existing technology, it has the advantages of specific site recognition, DNA catalytic cleavage, and high cleavage efficiency.

[0053] In other embodiments, other low-dimensional nanomaterials with defects can be used to replace molybdenum disulfide nanosheets to perform DNA site-specific cleavage.

[0054] The above description further details the present invention in conjunction with specific / preferred embodiments, and the specific implementation of the present invention should not be construed as being limited to these descriptions. Persons skilled in the art will appreciate that, without departing from the spirit of the present invention, they may make various substitutions or modifications to the described embodiments, and these substitutions or modifications should be considered to fall within the scope of protection of the present invention. Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "preferred embodiments," "examples," "specific examples," or "some examples" indicates that the specific features, structures, materials, or characteristics described in conjunction with such embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Persons skilled in the art may combine and assemble the different embodiments or examples described in this specification, as well as features of different embodiments or examples, without conflicting opinions. 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 may 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: A low-dimensional nanomaterial solution is mixed with a DNA solution, so as to utilize the catalytic activity of the low-dimensional nanomaterial to recognize and shear a specific site of the DNA; wherein the DNA has a specific site consisting of at least 7 consecutive bases T.

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 low-dimensional nanomaterial is at least one of a two-dimensional nanomaterial, a one-dimensional nanomaterial, and a zero-dimensional nanomaterial; preferably, the low-dimensional nanomaterial has at least one of a point defect, a line defect, and a surface defect.

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 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.

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 low-dimensional nanomaterial is molybdenum disulfide nanosheets.

5. The use of the low-dimensional nanomaterial as claimed in claim 1 in recognition and shearing of DNA specific sites, characterized in that: The DNA is pBR322 plasmid.

6. 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.

7. 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.

8. The use of the low-dimensional nanomaterial as claimed in claim 4 in recognition and shearing of DNA specific sites, characterized in that: 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 milling time is 150-300 minutes.

9. The use of the low-dimensional nanomaterial as claimed in claim 4 in recognition and shearing of DNA specific sites, characterized in that: The thickness of the molybdenum disulfide nanosheet is between 0.6nm and 20nm.

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