DNA hydrogel and preparation method thereof

Abstract

The invention discloses DNA hydrogel and a preparation method thereof. The preparation method comprises the following steps: designing and synthesizing a virtual gene containing three target DNA chain nucleotide sequences and an I-R3 deoxyribozyme nucleotide sequence; inserting the virtual gene into a plasmid vector to obtain a recombinant plasmid; transforming the recombinant plasmids into competent cells, and carrying out auxiliary amplification through auxiliary bacteriophages to obtain bacteriophage particles; splitting the bacteriophage particles to obtain precursor single-stranded DNA; adding the precursor single-stranded DNA into a first buffer solution containing zinc ions for self-cutting to obtain three target DNA chains; adding the three target DNA chains into a second buffer solution for assembling to obtain a precursor solution containing a Y structure unit; adjusting the precursor solution to be acidic to obtain the DNA hydrogel. The preparation method disclosed by the invention does not involve chemical synthesis and pollution waste discharge, is relatively low in cost and can be applied to large-scale production of the DNA hydrogel.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a DNA hydrogel and a preparation method thereof. Background Art

[0002] DNA hydrogels have attracted widespread attention due to their unique functions and sequence-dependent responsive properties. They have broad application prospects in the fields of cell-free protein expression, target cell capture, and ultra-soft robotics.

[0003] However, current DNA hydrogels synthesize DNA chains through solid-phase chemistry and require further purification to obtain high-purity target DNA chains. This is costly, low-quality, and complex, and produces chemically contaminated waste. The defects of the preparation method limit the large-scale production and application of DNA hydrogels.

[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 purpose of the present invention is to provide a DNA hydrogel and a preparation method thereof, aiming to solve the problems of the existing DNA hydrogel preparation method having high cost, low quality, complex process and generation of chemically contaminated waste.

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

[0007] The first aspect of the present invention provides a method for preparing a DNA hydrogel, comprising the steps of:

[0008] Designing and synthesizing a virtual gene comprising a first target DNA chain nucleotide sequence, a second target DNA chain nucleotide sequence, a third target DNA chain nucleotide sequence, and an I-R3 deoxyribozyme nucleotide sequence;

[0009] Inserting the virtual gene into a plasmid vector to obtain a recombinant plasmid;

[0010] The recombinant plasmid is transformed into competent cells, and a helper phage is added to assist amplification to obtain phage particles containing precursor single-stranded DNA;

[0011] Lysing the phage particles to obtain precursor single-stranded DNA;

[0012] Adding the precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage to obtain a first target DNA chain, a second target DNA chain, and a third target DNA chain;

[0013] Adding the first target DNA strand, the second target DNA strand, and the third target DNA strand into a second buffer for assembly to obtain a precursor solution containing a Y structural unit;

[0014] Adjusting the precursor solution to be acidic so as to cross-link the Y structural units in the precursor solution to obtain a DNA hydrogel;

[0015] Among them, the nucleotide sequence of the first target DNA chain is shown as SEQ ID NO.1, the nucleotide sequence of the second target DNA chain is shown as SEQ ID NO.2, the nucleotide sequence of the third target DNA chain is shown as SEQ ID NO.3, the nucleotide sequence of the virtual gene is shown as SEQ ID NO.4, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO.5.

[0016] In a preferred technical solution, the competent cells are selected from one of JM109 competent cells, DH5α competent cells, Top10 competent cells, and XL1-Blue competent cells.

[0017] In a preferred technical solution, the helper phage is selected from one of M13KO7, VCSM13, and AbMole super helper phage.

[0018] In a preferred technical solution, the first buffer solution containing zinc ions comprises 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, zinc acetate and water.

[0019] In a preferred technical solution, the second buffer comprises 4-morpholineethanesulfonic acid, sodium chloride and water.

[0020] According to a preferred technical solution, in the first buffer solution containing zinc ions, the concentration of the zinc ions is 0.4 to 3.2 mM.

[0021] According to a preferred technical solution, in the step of adding the precursor single-stranded DNA to a first buffer solution containing zinc ions for self-cleavage, the added concentration of the precursor single-stranded DNA is 100 to 1200 nM.

[0022] According to a preferred technical solution, in the precursor solution, the concentration of the Y structural unit is 0.5-2 mM.

[0023] According to a preferred technical solution, the reaction temperature of the self-cutting is 25 to 50° C., and the reaction time is 1 to 36 hours.

[0024] The second aspect of the present invention provides a DNA hydrogel prepared by the preparation method as described above.

[0025] Beneficial effects: Compared with the existing technology, the preparation method of the present invention has the following advantages:

[0026] (1) It does not involve chemical synthesis and polluting waste discharge, significantly reducing pollution in the production process, and is a green production technology;

[0027] (2) It does not involve expensive raw materials and biological enzymes, and the equipment used in the production process are all commonly used equipment in existing bioengineering, which greatly reduces the cost and can be applied to large-scale production;

[0028] (3) The biological method is used for DNA amplification, which has high sequence fidelity and produces target DNA chains of higher quality than those produced by traditional chemical synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart of the preparation of DNA hydrogel.

[0030] Figure 2 This is the DNA nucleotide sequence diagram of the Y structural unit.

[0031] Figure 3 This is a schematic diagram of obtaining the first target DNA chain based on self-cleavage of I-R3 DNAzyme.

[0032] Figure 4 It is an agarose gel electrophoresis diagram of the recombinant plasmid, its cleavage products and precursor single-stranded DNA.

[0033] Figure 5 It is an agarose gel electrophoresis diagram of the self-cleavage products of the precursor single-stranded DNA.

[0034] Figure 6 This is a diagram of the state changes of DNA hydrogel at different pH levels.

[0035] Figure 7 This is a scanning electron micrograph of a cross-section of a DNA hydrogel. DETAILED DESCRIPTION

[0036] The present invention provides a DNA hydrogel and a preparation method thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0037] The present invention provides a method for preparing a DNA hydrogel, comprising the steps of:

[0038] Designing and synthesizing a virtual gene comprising a first target DNA chain nucleotide sequence, a second target DNA chain nucleotide sequence, a third target DNA chain nucleotide sequence, and an I-R3 deoxyribozyme nucleotide sequence;

[0039] Inserting the virtual gene into a plasmid vector to obtain a recombinant plasmid;

[0040] The recombinant plasmid is transformed into competent cells, and a helper phage is added to assist amplification to obtain phage particles containing precursor single-stranded DNA;

[0041] Lysing the phage particles to obtain precursor single-stranded DNA;

[0042] Adding the precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage to obtain a first target DNA chain, a second target DNA chain, and a third target DNA chain;

[0043] Adding the first target DNA strand, the second target DNA strand, and the third target DNA strand into a second buffer for assembly to obtain a precursor solution containing a Y structural unit;

[0044] Adjusting the precursor solution to be acidic so as to cross-link the Y structural units in the precursor solution to obtain a DNA hydrogel;

[0045] Among them, the nucleotide sequence of the first target DNA chain is shown as SEQ ID NO.1, the nucleotide sequence of the second target DNA chain is shown as SEQ ID NO.2, the nucleotide sequence of the third target DNA chain is shown as SEQ ID NO.3, the nucleotide sequence of the virtual gene is shown as SEQ ID NO.4, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO.5.

[0046] The flow chart of the preparation method of the DNA hydrogel of the present invention is as follows Figure 1 Specifically, the present invention inserts three target DNA chains into two I-R3 deoxyribozymes respectively and connects them to obtain a virtual gene; then clones the virtual gene into a plasmid vector to obtain a recombinant plasmid containing the virtual gene; amplifies the recombinant plasmid with the assistance of a helper phage in a competent cell to obtain a phage particle containing a precursor single-stranded DNA, and then cleaves the phage particle to obtain a precursor single-stranded DNA; the precursor single-stranded DNA contains an I-R3 deoxyribozyme, which is a zinc ion-dependent self-cleaving deoxyribozyme. When the auxiliary factor zinc ion is present, it can specifically recognize the "GTTGAAAG" sequence to self-cleave ( Figure 2 ), therefore, in the first buffer containing zinc ions, the precursor single-stranded DNA is cut to generate the first target DNA chain (pH-Y3-1), the second target DNA chain (pH-Y3-2) and the third target DNA chain (pH-Y3-3); the three target DNA chains are assembled into a Y structural unit ( Figure 3); Under acidic conditions, the response sequence (CCCCTAACCCC) at the end of a Y structural unit branch will form a four-stranded non-covalent complex with two double strands inserted in antiparallel order with the response sequence at the end of another Y structural unit branch, namely the i-motif structure, so that multiple Y structural units can be assembled to form a DNA hydrogel with pH-responsive properties. When the environment of the DNA hydrogel changes from acidic to alkaline, the i-motif structure will be destroyed, causing the DNA hydrogel to turn into a solution again.

[0047] In some embodiments, the competent cells are selected from one of JM109 competent cells, DH5α competent cells, Top10 competent cells, and XL1-Blue competent cells, but are not limited thereto. Preferably, the competent cells are JM109 competent cells.

[0048] In some embodiments, the helper phage is selected from one of M13KO7, VCSM13, and AbMole super helper phage, but is not limited thereto. Preferably, the helper phage is M13KO7.

[0049] In some embodiments, the first buffer containing zinc ions includes 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), sodium chloride, zinc acetate and water, but is not limited thereto. Preferably, in the first buffer containing zinc ions, the concentration of HEPES is 100 mM, the concentration of sodium chloride is 200 mM, and the concentration of zinc acetate is 4 mM; the pH of the first buffer containing zinc ions is 7.

[0050] In some embodiments, the second buffer comprises 4-morpholineethanesulfonic acid (MES), sodium chloride and water, but is not limited thereto. Preferably, in the second buffer, the concentration of MES is 50 mM, the concentration of sodium chloride is 50 mM; and the pH of the second buffer is 8.

[0051] In some embodiments, in the first buffer containing zinc ions, the concentration of zinc ions is 0.4 to 3.2 mM, but is not limited thereto. Preferably, the concentration of zinc ions is 2 to 2.8 mM. Specifically, when the concentration of zinc ions is lower than 0.4 mM, I-R3 deoxyribozyme cannot be cut; when the concentration of zinc ions is higher than 2.8 mM, the concentration of precursor single-stranded DNA is limited, which will lead to a decrease in cutting efficiency.

[0052] In some embodiments, in the step of adding the precursor single-stranded DNA to the first buffer containing zinc ions for self-cleavage, the concentration of the added precursor single-stranded DNA is 100 to 1200 nM, but is not limited thereto. Preferably, the added concentration of the precursor single-stranded DNA is 450 to 900 nM. Specifically, the precursor single-stranded DNA will gel at a high concentration, and controlling the added concentration to be lower is conducive to improving the complete cleavage efficiency.

[0053] In some embodiments, in the precursor solution, the concentration of the Y structural unit is 0.5-2 mM, but not limited thereto. Preferably, the concentration of the Y structural unit is 0.8-1.2 mM; more preferably, the concentration of the Y structural unit is 1 mM.

[0054] In some embodiments, the step of lysing the phage particles adopts an alkaline lysis method.

[0055] In some embodiments, the reaction temperature of the self-cleavage is 25-50°C, and the reaction time is 1-36 hours, but not limited thereto. Preferably, the reaction temperature of the self-cleavage is 37°C, and the reaction time of the self-cleavage is related to the concentration of zinc ions and the concentration of the precursor single-stranded DNA, and can be adjusted according to actual conditions.

[0056] In some embodiments, the specific steps of assembling include:

[0057] adding the first target DNA strand, the second target DNA strand, and the third target DNA strand into a second buffer to obtain a mixed solution;

[0058] The mixed solution was heated to 95°C for 5 min and then cooled to 25°C within 2 hours.

[0059] In some embodiments, the acidity refers to a pH of 4 to 6.5, but is not limited thereto. Preferably, the acidity refers to a pH of 5.

[0060] The embodiment of the present invention also provides a DNA hydrogel, which is prepared by the preparation method as described above.

[0061] In some embodiments, the DNA hydrogel transforms into a solution under alkaline conditions.

[0062] The present invention will be further described below by means of specific examples.

[0063] Example 1

[0064] This embodiment provides a DNA hydrogel, and the preparation method thereof is as follows:

[0065] The first target DNA chain, the second target DNA chain, and the third target DNA chain were respectively inserted between two I-R3 deoxyribozymes and connected to obtain virtual genes; then the virtual genes were cloned into a customized plasmid vector with an ampicillin resistance gene to obtain a recombinant plasmid containing the virtual gene. The recombinant plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and sequenced and verified.

[0066] Among them, the nucleotide sequence of the first target DNA chain is shown as SEQ ID NO.1, the nucleotide sequence of the second target DNA chain is shown as SEQ ID NO.2, the nucleotide sequence of the third target DNA chain is shown as SEQ ID NO.3, the nucleotide sequence of the virtual gene is shown as SEQ ID NO.4, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO.5.

[0067] The constructed recombinant plasmid was transformed into JM109 competent cells by heat shock. In a 250 mL shake flask, a fresh clone was selected and added to 50 mL of 2×YT medium containing 5 mM MgCl2 and 50 μg / mL ampicillin. The cells were cultured at 37°C and 200 rpm for 2 hours until slightly turbid (A600 < 0.05, < 10 Klett). 50 μL of M13KO7 helper phage (1×10 11 pfu / mL), the final concentration was 1×10 8 pfu / mL, then cultured at 37°C, 200rpm for 90 minutes. Kanamycin was added to the culture medium to a final concentration of 70 μg / mL, and then the bacteria were cultured overnight at 37°C, 250rpm. The bacteria were precipitated by centrifugation at 12000g for 10 minutes, and then 10mL of 2.5M NaCl / 20% PEG-8000 was added to 50mL of the supernatant to precipitate the phage particles in the supernatant, and centrifuged at 12000g for 10 minutes. The phage particles were resuspended in 5mL 1×TE buffer (10mMTris, 1mM EDTA, pH=8), and then centrifuged at 12000g for 10 minutes again to precipitate any remaining cells. The precursor single-stranded DNA was isolated from the phage particles using alkaline lysis.

[0068] The constructed recombinant plasmid was verified by single and double enzyme digestion, and characterized by GelRed pre-stained agarose gel electrophoresis with the prepared precursor single-stranded DNA. After loading the sample, it was run on a 1.2% agarose gel in 1×TAE buffer at 110V for 45 minutes, and then imaged on a Fujifilm LAS-3000Imager (Fuji Photo Film, Japan). The results are shown in Figure 4 shown.

[0069] Figure 4 In the figure, lane 1 is a DNA size marker; lane 2 is a recombinant plasmid containing a virtual gene; lane 3 is a linearized recombinant plasmid obtained by single enzyme digestion; lane 4 is a virtual gene (bottom) and a plasmid vector (top) separated by double enzyme digestion; lane 5 is a precursor single-stranded DNA.

[0070] 2× buffer was prepared by dissolving 4 mM zinc acetate in a buffer containing 100 mM HEPES and 200 mM NaCl, with a final pH of 7. The self-cleavage product obtained by mixing the 2× buffer with the precursor single-stranded DNA and incubating at 37°C for 36 hours contained the first target DNA strand, the second target DNA strand, and the third target DNA strand. The self-cleavage product was precipitated with 75% cold ethanol and quantified on NanoDrop One (Thermo Scientific, USA).

[0071] The first target DNA chain, the second target DNA chain, and the third target DNA chain produced by chemical synthesis and biotechnology were dissolved in 1×TAE / Mg 2+ Buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, and 12.5 mM Mg 2+ ) and incubated at 95°C for 5 minutes, the solution was slowly cooled to room temperature over 2 hours. 2+ The gel was removed and stained with GelRed and imaged on a Fujifilm LAS-3000Imager (Fuji Photo Film, Japan). Figure 5 shown.

[0072] Figure 5 In the figure, lane 1 is a DNA size marker; lane 2 is a chemically synthesized first target DNA chain; lane 3 is an assembly of the chemically synthesized first target DNA chain and the second target DNA chain; lane 4 is a Y structural unit assembled from the chemically synthesized first target DNA chain, the second target DNA chain and the third target DNA chain; lane 5 is a self-cleavage product of the precursor single-stranded DNA.

[0073] The self-cleavage product of the precursor single-stranded DNA was dissolved in 200 μL of 50 mM MES buffer (pH 8.0) containing 50 mM NaCl, and the Y structural unit was formed by an annealing procedure of heating to 95°C and then slowly cooling to room temperature within 2 hours to obtain a precursor solution. The concentration of the Y structural unit in the precursor solution was 1 mM. 5 μL of 6M HCl was added to the precursor solution to adjust the pH value to 5.0, at which time the solution turned into a gel, and 5 μL of 6M NaOH was added to adjust the pH value to 8.0, at which time the gel turned back into a solution. The results of this process are shown in Figure 6 The cross-sectional scanning electron micrograph of the DNA hydrogel prepared at pH 5.0 is shown in Figure 7 shown.

[0074] In summary, the present invention provides a DNA hydrogel and a preparation method thereof, the preparation method comprising the steps of: designing and synthesizing a virtual gene comprising a first target DNA chain nucleotide sequence, a second target DNA chain nucleotide sequence, a third target DNA chain nucleotide sequence and an I-R3 deoxyribozyme nucleotide sequence; inserting the virtual gene into a plasmid vector to obtain a recombinant plasmid; transforming the recombinant plasmid into competent cells, adding a helper phage to assist in amplification, and obtaining phage particles comprising a precursor single-stranded DNA; lysing the phage particles to obtain a precursor single-stranded DNA; adding the precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage to obtain a first target DNA chain, a second target DNA chain, and a third target DNA chain; adding the first target DNA chain, the second target DNA chain, and the third target DNA chain to a second buffer for assembly to obtain a precursor solution comprising a Y structural unit; adjusting the precursor solution to acidity to cross-link the Y structural unit in the precursor solution to obtain a DNA hydrogel; wherein the nucleotide sequence of the first target DNA chain is as shown in SEQ ID NO.1, and the nucleotide sequence of the second target DNA chain is as shown in SEQ ID The nucleotide sequence of the third target DNA chain is shown in SEQ ID NO.2, the nucleotide sequence of the virtual gene is shown in SEQ ID NO.3, the nucleotide sequence of the virtual gene is shown in SEQ ID NO.4, and the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.5.

[0075] The preparation method of the present invention does not involve chemical synthesis and polluting waste discharge, significantly reduces pollution in the production process, and is a green production technology; it does not involve expensive raw materials and biological enzymes, and the equipment and devices used in the production process are all commonly used equipment in existing bioengineering, which greatly reduces the cost and can be applied to large-scale production; in addition, the biological method is used for DNA amplification, the sequence fidelity is high, and the quality of the target DNA chain produced is higher than that of the product of traditional chemical synthesis.

[0076] 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 preparing a DNA hydrogel, characterized in that: Includes steps: Designing and synthesizing a virtual gene comprising a first target DNA chain nucleotide sequence, a second target DNA chain nucleotide sequence, a third target DNA chain nucleotide sequence, and an I-R3 deoxyribozyme nucleotide sequence; Inserting the virtual gene into a plasmid vector to obtain a recombinant plasmid; The recombinant plasmid is transformed into competent cells, and a helper phage is added to assist amplification to obtain phage particles containing precursor single-stranded DNA; Lysing the phage particles to obtain precursor single-stranded DNA; Adding the precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage to obtain a first target DNA chain, a second target DNA chain, and a third target DNA chain; Adding the first target DNA strand, the second target DNA strand, and the third target DNA strand into a second buffer for assembly to obtain a precursor solution containing a Y structural unit; Adjusting the precursor solution to be acidic so as to cross-link the Y structural units in the precursor solution to obtain a DNA hydrogel; Among them, the nucleotide sequence of the first target DNA chain is shown as SEQ ID NO.1, the nucleotide sequence of the second target DNA chain is shown as SEQ ID NO.2, the nucleotide sequence of the third target DNA chain is shown as SEQ ID NO.3, the nucleotide sequence of the virtual gene is shown as SEQ ID NO.4, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO.

5.

2. The preparation method according to claim 1, characterized in that: The competent cells are selected from one of JM109 competent cells, DH5α competent cells, Top10 competent cells, and XL1-Blue competent cells.

3. The preparation method according to claim 1, characterized in that: The helper phage is selected from one of M13KO7, VCSM13, and AbMole super helper phage.

4. The preparation method according to claim 1, characterized in that: The first buffer containing zinc ions comprises 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, zinc acetate and water.

5. The preparation method according to claim 1, characterized in that: The second buffer comprises 4-morpholineethanesulfonic acid, sodium chloride and water.

6. The preparation method according to claim 1, characterized in that: In the first buffer solution containing zinc ions, the concentration of the zinc ions is 0.4-3.2 mM.

7. The preparation method according to claim 1, characterized in that: In the step of adding the precursor single-stranded DNA to a first buffer containing zinc ions for self-cleavage, the added concentration of the precursor single-stranded DNA is 100-1200 nM.

8. The preparation method according to claim 1, characterized in that: In the precursor solution, the concentration of the Y structural unit is 0.5-2 mM.

9. The preparation method according to claim 1, characterized in that: The reaction temperature of the self-cutting is 25-50° C., and the reaction time is 1-36 hours.

10. A DNA hydrogel, characterized in that: The preparation is obtained by the preparation method according to any one of claims 1 to 9.

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