Method for shearing DNA

Abstract

The invention relates to the field of gene editing, in particular to a method for shearing DNA. The method for shearing the DNA comprises the following steps: mixing and incubating amino polystyrene nano-microspheres and DNA under a dark condition, so as to obtain the sheared DNA. The method disclosed by the invention can be realized under a dark condition without special illumination, the condition is simple, the cost is low, the DNA shearing effect of the amino polystyrene nano-microspheres under different temperature and pH conditions is excellent, and the environmental tolerance is high. The method disclosed by the invention can also be converted into a method for turning waste into wealth, DNA is cut by utilizing nano plastic in a polluted water environment, microorganisms in sewage are treated, and the method has important guiding significance on sewage treatment.

Description

Technical Field

[0001] The present invention relates to the field of gene editing, and in particular to a method for shearing DNA. Background Art

[0002] There are many measures to prevent and control microbial contamination in water environments, such as the use of oxidants such as hydrogen peroxide (H 2 O 2 ) can damage DNA by generating free radicals. Some DNA crosslinkers, such as propane sulfonate and nitrogen mustard compounds, can react with DNA bases, resulting in crosslinks between or within DNA chains, affecting DNA replication and transcription. However, these methods often cause secondary pollution to the aquatic environment.

[0003] Studies have found that nanomaterials have a good effect on shearing DNA. On the one hand, there are already some nanomaterials that can be used to shear DNA, such as graphene oxide, carbon nanotubes, molybdenum disulfide, etc., but the preparation methods of these nanomaterials are complicated and the preparation cost is high. On the other hand, there are also existing methods for shearing DNA using nanospheres, but they require specific light (UV) to occur, the conditions are harsh and the cost is high (DNA cleavage and chemical transformation of nano-plastics mediated by surface ligand and Size. Chem. Commun., 2021, 57, 9740. DOI: 10.1039 / d1cc04028f).

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for shearing DNA, aiming to solve the problem that the existing methods for shearing DNA have harsh conditions and high costs.

[0006] The technical solution of the present invention is as follows:

[0007] The invention provides a method for shearing DNA, wherein the method comprises: incubating amino polystyrene nanospheres and DNA in a mixed manner under dark conditions to obtain sheared DNA.

[0008] Optionally, the method specifically includes:

[0009] dispersing amino polystyrene nano-microspheres in water to obtain an amino polystyrene nano-microsphere dispersion;

[0010] Dissolve the DNA in PBS buffer to obtain DNA buffer;

[0011] In the dark, the amino polystyrene nanosphere dispersion and the DNA buffer solution are mixed and incubated for 0.5h-3h to obtain the sheared DNA.

[0012] Optionally, the DNA is pBR322 DNA.

[0013] Optionally, the particle size of the amino polystyrene nanospheres is 1 nm to 1000 nm.

[0014] Optionally, the mass ratio of the amino polystyrene nanospheres to DNA is 5:3-50.

[0015] Optionally, the incubation temperature is 4°C to 44°C, and the incubation pH is 5.0 to 9.0.

[0016] Optionally, the concentration of amino polystyrene nano-microspheres in the amino polystyrene nano-microsphere dispersion is 10 mg / L to 200 mg / L.

[0017] Optionally, the concentration of DNA in the DNA buffer is 0.003ug / uL to 0.015ug / uL.

[0018] Beneficial effects: The present invention provides a method for shearing DNA using specific amino polystyrene nano-microspheres. Compared with the existing methods for shearing DNA, the method of the present invention can be implemented in dark conditions without special illumination, with simple conditions and low cost. In addition, the amino polystyrene nano-microspheres have excellent effects on shearing DNA under different temperatures and different pH conditions, and have high environmental tolerance. In addition, the amino polystyrene nano-microspheres used in the method of the present invention are a kind of nano plastic, so they can be converted into a method of turning waste into treasure. By using nano plastics in a polluted water environment to cut DNA, the microorganisms in sewage can be treated, which has important guiding significance for sewage treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The gel electrophoresis diagram of the DNA of Example 1-4.

[0020] Figure 2 This is the gel electrophoresis diagram of the DNA of Comparative Example 1-4.

[0021] Figure 3 This is the gel electrophoresis diagram of the DNA of Example 5-7.

[0022] Figure 4 The gel electrophoresis diagram of the DNA of Examples 8-12.

[0023] Figure 5 This is the gel electrophoresis diagram of the DNA of Examples 13-15.

[0024] Figure 6 This is a structural model diagram of the pBR322 DNA molecule.

[0025] Figure 7 For LPS-NH 2 Diagram of the structural model of a molecule.

[0026] Figure 8 This is the structural model diagram of the LPS-COOH molecule.

[0027] Fig. 9 These are the conformational diagrams of the simulation system 1 at different simulation times.

[0028] Fig.10 To simulate the LPS-NH 2 Coordinate diagram of the interaction energy between the molecule and the DNA molecule at different simulation times.

[0029] Fig.11 It is the conformation diagram of simulation system 2 at different simulation times.

[0030] Fig.12 This is the interaction energy coordinate diagram between LPS-COOH molecules and DNA molecules in simulation system 2 at different simulation times. DETAILED DESCRIPTION

[0031] The present invention provides a method for shearing DNA. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] According to one embodiment of the present invention, a method for shearing DNA is provided, wherein the method comprises: incubating amino polystyrene nanospheres and DNA in a mixed manner under dark conditions to obtain sheared DNA.

[0033] The embodiment of the present invention utilizes specific amino polystyrene nanoparticles and DNA for mixed incubation, and can achieve DNA shearing in the dark, with simple conditions and low cost. The structural formula of the amino polystyrene used in the embodiment of the present invention is This type of group-modified nanoplastic is more common in some polluted waters. Therefore, the embodiment of the present invention is a method of turning waste into treasure. It can use nanoplastics in polluted water environments to cut microbial DNA, thereby treating microorganisms in sewage. It has the advantages of low acquisition cost, simple reaction conditions and easy implementation.

[0034] The embodiment of the present invention utilizes the chemical properties of specific groups in amino polystyrene nano-microspheres to shear DNA. There are electrostatic interaction energy and van der Waals interaction energy between amino groups and DNA molecules, so amino polystyrene nano-microspheres can effectively shear DNA molecules.

[0035] According to one embodiment of the present invention, a method for shearing DNA is provided, the method specifically comprising:

[0036] dispersing amino polystyrene nano-microspheres in water to obtain an amino polystyrene nano-microsphere dispersion;

[0037] Dissolve the DNA in PBS buffer to obtain DNA buffer;

[0038] In the dark, the amino polystyrene nanosphere dispersion and the DNA buffer solution are mixed and incubated for 0.5h-3h to obtain the sheared DNA.

[0039] According to one embodiment of the present invention, the PBS buffer is a standard PBS buffer with a pH of 7.4 and a 0.1 M buffer.

[0040] According to one embodiment of the present invention, the DNA is pBR322 DNA.

[0041] The pBR322 DNA in the present invention is a widely used and studied standard plasmid, whose detailed sequence and structural information are public, and whose size is moderate, with a length of about 4.4 kb, which is convenient for operation and analysis in experiments. This plasmid is relatively stable in replication and maintenance in bacteria, easy to amplify and purify, and is suitable for standardized operation of DNA damage experiments, making it an ideal model for studying DNA damage effects.

[0042] The present invention uses pBR322 DNA to study the shearing effect of nanoplastics on DNA. Since nanoplastics in sewage can easily enter microorganisms and accumulate for a long time, the discovery that amino polystyrene nanoplastics have the ability to shear DNA means that they also have the ability to destroy microbial DNA, thereby causing the death of microorganisms, thereby achieving the purpose of treating the water environment contaminated by microorganisms.

[0043] According to one embodiment of the present invention, the mixed incubation time is 0.5h-3h, for example, 0.5h, 1h, 2h, 3h, etc. As the incubation time increases, the shearing effect of the amino polystyrene nanospheres on DNA gradually increases.

[0044] According to one embodiment of the present invention, the particle size of the aminopolystyrene nanospheres is 1nm to 1000nm. Preferably, the particle size of the aminopolystyrene nanospheres is 10nm to 500nm; more preferably, the particle size of the aminopolystyrene nanospheres is 40nm to 200nm; for example, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 120nm, 150nm, 180nm, 200nm, etc. The particle size of the aminopolystyrene nanospheres cannot be too small or too large. In the specific embodiment of the present invention, the particle size of the aminopolystyrene nanospheres is 90nm, at which time it has the best shearing effect on DNA. The inventors also tested the shearing effect of the aminopolystyrene nanospheres with a particle size of 2000nm on DNA and found that they basically have no or very little ability to cut DNA.

[0045] According to one embodiment of the present invention, the mass ratio of the amino polystyrene nanospheres to DNA is 5:3 to 50. Within this mass ratio range, the amino polystyrene nanospheres have an excellent shearing effect on DNA. Preferably, the mass ratio of the amino polystyrene nanospheres to DNA is 5:3, in which case the amino polystyrene nanospheres have a particularly excellent shearing effect on DNA.

[0046] According to one embodiment of the present invention, the incubation temperature is 4°C-44°C, and the incubation pH is 5.0-9.0. Within the temperature and pH range, the amino polystyrene nanospheres show excellent shearing effect on DNA, indicating that the DNA shearing method of the present invention has wide universality and high environmental tolerance. Therefore, when the method of the present invention is applied to sewage treatment, it shows good adaptability under different external temperatures and different pH sewage environmental conditions.

[0047] According to one embodiment of the present invention, the concentration of the aminopolystyrene nanospheres in the aminopolystyrene nanosphere dispersion is 10 mg / L-200 mg / L, such as 10 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, etc. Within the range of 10 mg / L-200 mg / L, as the concentration of the aminopolystyrene nanospheres increases, the shearing effect on DNA gradually increases.

[0048] According to one embodiment of the present invention, the concentration of DNA in the DNA buffer is 0.003ug / uL to 0.015ug / uL. For example, it can be 0.003ug / uL, 0.004ug / uL, 0.005ug / uL, 0.006ug / uL, 0.007ug / uL, 0.008ug / uL, 0.009ug / uL, 0.010ug / uL, 0.011ug / uL, 0.012ug / uL, 0.013ug / uL, 0.014ug / uL, 0.015ug / uL, etc.

[0049] In a preferred embodiment, the concentration of DNA in the DNA buffer is 0.003 ug / uL. At this concentration, the shearing effect is the best.

[0050] The present invention will be further described below by means of specific examples.

[0051] In the following examples, pBR322 DNA was purchased from Thermo Fisher Scientific, gel loading dye was purchased from Beyotime Biotechnology, and all other chemicals were purchased from Aladdin and dissolved in deionized water after treatment with a milli-Q water purification system (Millipore, Billerica, MA, US).

[0052] Example 1

[0053] This embodiment provides a method for shearing DNA, which is as follows:

[0054] Amino polystyrene nanospheres (PS-NH 2 Nanoplastics, particle size 90nm) were dispersed in water to obtain 10mg / L aminopolystyrene nanosphere dispersion;

[0055] Dilute a 0.1M, pH=7.4 standard PBS buffer to 5mM, dissolve the DNA in the diluted PBS buffer to obtain a 0.003ug / uL DNA buffer (DNA is pBR322DNA);

[0056] In the dark, the amino polystyrene nanosphere dispersion and the DNA buffer were mixed and incubated at room temperature for 2 hours, wherein the pH was 7.4 and the mass ratio of the amino polystyrene nanospheres to the DNA was 5:3, to obtain the sheared DNA.

[0057] Embodiment 2-4

[0058] The method of shearing DNA is the same as that in Example 1, except that the concentration of the aminopolystyrene nanosphere dispersion is adjusted to 50 mg / L, 100 mg / L and 200 mg / L.

[0059] Comparative Examples 1-4

[0060] The method of shearing DNA is the same as that in Example 1, except that carboxyl polystyrene nanospheres are used to shear DNA. Carboxyl polystyrene nanospheres (PS-COOH nanoplastics for short, with a particle size of 90 nm) are dispersed in water to obtain 10 mg / L, 50 mg / L, 100 mg / L and 200 mg / L amino polystyrene nanosphere dispersions.

[0061] DNA detection was performed on the systems after incubation in the above-mentioned Examples 1-4 and Comparative Examples 1-4. The specific method was as follows: gel-loaded dye was added to the system after incubation, and the experiment was performed using a Beijing Liuyi Biological Agarose Horizontal Electrophoresis Instrument and a power adapter from the same manufacturer. The electrophoresis solution was diluted to 1×TAE using a standard 50×TAE (triacetate-EDTA) buffer, and 1% agarose gel was used. The system was powered on at 80V for 45 minutes to obtain the test results.

[0062] Figure 1 The gel electrophoresis diagram of the DNA of Example 1-4, wherein the Form I band represents the uncut pBR322 DNA (circular plasmid), and the Form II band represents the cut pBR322 DNA (open-circular state). It can be seen from the figure that with the PS-NH 2 As the concentration of nanoplastics increases, the Form II band gradually becomes darker, indicating that the shearing effect on DNA gradually increases.

[0063] Figure 2 The gel electrophoresis diagram of the DNA of Comparative Examples 1-4 shows that the PS-COOH nanoplastics at different concentrations have basically no shearing effect on the DNA.

[0064] Embodiment 5-7

[0065] The method of shearing DNA was the same as that in Example 1, except that the time of mixed incubation was adjusted to 0.5 h, 1 h and 3 h.

[0066] The same method as in Example 1 was used to perform DNA detection on the systems after incubation in Examples 5-7. Figure 3 This is the gel electrophoresis diagram of the DNA of Example 5-7. It can be seen from the figure that when the incubation time is 0.5h, the From II band begins to appear. When the incubation time increases from 1h to 3h, the From II band gradually changes from light to dark, indicating that the DNA shearing effect is gradually enhanced.

[0067] Examples 8-12

[0068] The method of shearing DNA is the same as that in Example 1, except that the pH during mixed incubation is adjusted to 5.0, 6.0, 7.0, 8.0 and 9.0.

[0069] The same method as in Example 1 was used to perform DNA detection on the systems after incubation in Examples 8-12. Figure 4 The gel electrophoresis diagram of the DNA of Examples 8-12 shows that under different pH conditions, amino polystyrene nanoparticles exhibit excellent shearing effects on DNA, which also shows that PS-NH 2 DNA shearing is not affected by the pH value of the aquatic environment.

[0070] Examples 13-15

[0071] The method of shearing DNA was the same as that in Example 1, except that the temperature during the mixed incubation was adjusted to 4°C, 24°C and 44°C.

[0072] The same method as in Example 1 was used to perform DNA detection on the systems after incubation in Examples 13-15. Figure 5 The gel electrophoresis diagram of the DNA of Examples 13-15 shows that under different temperature conditions, amino polystyrene nanoparticles show excellent shearing effect on DNA, which also shows that PS-NH 2 The shearing of DNA is not affected by temperature.

[0073] Example 16

[0074] In order to explore the mechanism of DNA shearing by amino polystyrene nanospheres of the present invention, a molecular dynamics simulation experiment was conducted. The software for molecular dynamics simulation was GROMACS2021, the molecular force field was GAFF force field, and two different sets of simulation systems were constructed:

[0075] Simulation system 1 contains a pBR322 DNA molecule (structure Figure 6 as shown) and 1 LPS-NH 2 molecules, in which LPS-NH 2 The molecule refers to a long-chain amino polystyrene polymer with a repeating unit of 50 and a molecular weight of 5960. 2 Molecular structure Figure 7 shown.

[0076] Simulation system 2 includes one pBR322 DNA molecule and one LPS-COOH molecule, wherein the LPS-COOH molecule refers to a long-chain carboxyl polystyrene polymer with 40 repeating units and a molecular weight of 5928. The molecular structure of LPS-COOH is as follows: Figure 8 shown.

[0077] Then, the two groups of simulation systems were solvated with water respectively, and the steep method was used to perform 50,000 steps of energy minimization on the two groups of simulation systems under the force convergence criterion of 1000 kJ / mol / nm. Then, NVT and NPT simulations were performed in turn with 100 ps of temperature (298.15 K) and pressure (1 atm) control, followed by 50 ns of molecular dynamics simulation to obtain the simulation results.

[0078] Fig. 9 The conformation diagrams of the simulation system 1 at different simulation times, where a, b, and c are the conformation diagrams at 0 ns, 30 ns, and 50 ns, respectively. The stick-shaped part represents LPS-NH 2 molecular. Fig.10 is the LPS-NH in simulation system 1 2 The interaction energy coordinate diagram between LPS-NH 2 There is interaction energy between the amino polystyrene nanoparticles and the DNA molecules, which proves that the amino polystyrene nanoparticles in the present invention can effectively shear the DNA molecules.

[0079] Fig.11 These are the conformational diagrams of the simulation system 2 at different simulation times, where a, b, and c are the conformational diagrams at 0 ns, 30 ns, and 50 ns, respectively, and the stick-shaped part represents the LPS-COOH molecule. Fig.12 This is the interaction energy coordinate diagram between LPS-COOH molecules and DNA molecules at different simulation times in simulation system 2. It can be seen that after about 10ns, there is basically no interaction energy between LPS-COOH molecules and DNA molecules, which means that LPS-COOH molecules cannot effectively cut DNA molecules.

[0080] In summary, in the method for shearing DNA provided by the present invention, amino polystyrene nanospheres can shear DNA well, which can be achieved in dark conditions, with simple conditions and low cost, and is insensitive to temperature and pH and has high environmental tolerance.

[0081] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for shearing DNA, characterized in that: The method comprises: incubating the amino polystyrene nanospheres and DNA in a mixed manner under dark conditions to obtain sheared DNA.

2. A method for shearing DNA according to claim 1, characterized in that: The method specifically comprises: dispersing amino polystyrene nano-microspheres in water to obtain an amino polystyrene nano-microsphere dispersion; Dissolve the DNA in PBS buffer to obtain DNA buffer; In the dark, the amino polystyrene nanosphere dispersion and the DNA buffer solution are mixed and incubated for 0.5h-3h to obtain the sheared DNA.

3. A method for shearing DNA according to claim 1 or 2, characterized in that: The DNA is pBR322 DNA.

4. A method for shearing DNA according to claim 1 or 2, characterized in that: The particle size of the amino polystyrene nanospheres is 1nm to 1000nm.

5. A method for shearing DNA according to claim 1 or 2, characterized in that: The mass ratio of the amino polystyrene nanospheres to the DNA is 5:3-50. 。 6. A method for shearing DNA according to claim 1 or 2, characterized in that: The incubation temperature is 4° C. to 44° C., and the incubation pH is 5.0 to 9.

0.

7. A method for shearing DNA according to claim 2, characterized in that: The concentration of the amino polystyrene nano microspheres in the amino polystyrene nano microsphere dispersion is 10 mg / L to 200 mg / L.

8. A method for shearing DNA according to claim 2, characterized in that: The concentration of DNA in the DNA buffer is 0.003ug / uL to 0.015ug / uL.

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