A method for preparing hydrogel based on the activity of phi29 DNA polymerase rolling circle replication of long-chain base-modified nucleic acid

Abstract

The present invention discloses a method for preparing a hydrogel based on the activity of phi29 DNA polymerase rolling circle replication of long-chain base-modified nucleic acids, belonging to the technical field of biomaterials. The method of the present invention is operated as follows: In a rolling circle amplification reaction system containing phi29 DNA polymerase, a pairable artificial nucleotide with an alkynylamino modification at the 5th position of the base part is incorporated to prepare a nucleic acid with an amino linker on some bases; the obtained nucleic acid is concentrated and annealed, and the annealed product is mixed with a chemical crosslinking agent in a phosphate buffer solution and incubated to obtain the hydrogel. The present invention synthesizes nucleic acids with modified bases by an enzymatic method, provides covalent binding sites, and solves the problem of low stability of DNA hydrogels based on hydrogen bond binding by introducing covalent bonds. A large amount of nucleic acids can be quickly obtained through rolling circle amplification, providing a simple and highly universal production method for the formation of novel nucleic acid hydrogels.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to a method for preparing a hydrogel based on the activity of phi29 DNA polymerase rolling circle replication of long-chain base-modified nucleic acids. Background Art

[0002] Phi29 DNA polymerase is a thermophilic (30 °C) DNA polymerase derived from the Bacillus subtilis phage phi29 and belongs to the B-type DNA polymerase [1] . In 2001, phi29 DNA polymerase was first used for isothermal multiple primer rolling circle amplification [2] . Utilizing the special strand displacement and continuous synthesis characteristics of phi29 DNA polymerase, with circular DNA as the template and short DNA complementary to part of the circular template as the primer, single-stranded DNA is synthesized using dNTPs as raw materials under the catalysis of phi29 DNA polymerase. The single-stranded DNA product contains nucleic acid fragments with multiple tandem repeats and can be formed without the thermal cycling steps required for conventional PCR. The isothermal multiple primer rolling circle amplification method is characterized by high speed and efficiency, capable of achieving a 10,000-fold amplification within a short time and continuously synthesizing DNA fragments up to 70 kb in length. At the same time, phi29 DNA polymerase has a 3' to 5' exonuclease proofreading function, so the error rate is 1000 times lower than that of the standard Taq polymerase [3] . The amplification products can be used for SNP genotyping, DNA sequencing, etc. The multi-primer rolling circle amplification using phi29 DNA polymerase also has the advantage of being able to produce double-stranded products, making it possible to apply it to subsequent restriction endonuclease digestion or other related methods of gene cloning, nucleic acid labeling, and target detection.

[0003] Previous studies have explored the activity of phi29 DNA polymerase in recognizing partially sugar-modified nucleic acids. Hexose nucleic acid (HNA) and arabinose nucleic acid (FANA) do not have obvious steric hindrance in the active site of this polymerase. The exonuclease-deficient phi29 DNA polymerase exhibits synthetic activity towards such XNAs and may achieve rolling circle amplification to synthesize long-chain nucleic acid molecules containing such XNAs [4]. In recent years, due to these unique properties, phi29 DNA polymerase has been widely used in the preparation of various DNA-functional polymers. Among them, DNA hydrogel is a cross-linked hydrophilic polymer, a three-dimensional polymer network constructed with DNA as the structural unit, and has become an international hot research direction in recent years. DNA hydrogel not only utilizes the framework structure of hydrogel but also retains the biological functions of DNA, achieving a perfect integration of the structure and function of hydrogel materials, and showing broad application prospects in the fields of biosensor preparation, drug delivery, cell culture, in vitro protein synthesis, intelligent device development, environmental protection, etc. In addition, with the emergence of precision medicine and molecular diagnostic technologies, phi29 DNA polymerase will have important application value in nucleic acid sequencing and virus detection. DNA is the core genetic material of the life system, guiding biological development and the operation of life functions. From the perspective of material chemistry, DNA is a natural biopolymer with characteristics that cannot be compared with synthetic polymers. For example: the base complementary pairing property enables DNA to have precise and efficient self-assembly ability; the DNA sequence is diverse and adjustable, the structure is precisely controllable, and it has rich stimulus responsiveness; natural evolution endows organisms with a rich variety of biological enzymes that can precisely operate on DNA at the molecular level; DNA has good biocompatibility and biodegradability.

[0004] Most base-modified nucleic acids are derivatives formed by chemically modifying different parts of natural bases. Compared with natural nucleic acids, some base-modified nucleic acids have more abundant properties and great application potential in the fields of bioengineering, nanotechnology, molecular biology, and medicine. There are mainly two methods for synthesizing base-modified nucleic acids: chemical synthesis and enzymatic synthesis. Usually, the chemical synthesis of base-modified nucleic acids is difficult to operate and inefficient. In addition, some modifications cannot be introduced by chemical methods. Enzymatic synthesis is another method for preparing base-modified nucleic acids. At present, the synthesis of base-modified nucleic acids containing 5-substituted pyrimidine analogs [5-6] and various purine derivatives by polymerase chain reaction (PCR) has been reported. [7] . However, this type of synthesis method relies on cumbersome thermal cycling steps, is inefficient, and cannot uniformly amplify the products, greatly limiting the popularization and application of base-modified nucleic acids.

[0005] References:

[0006] [1] Arunas L, Zivile K, Vilma Z-R, et al. Duality of polynucleotide substrates for Phi29 DNA polymerase: 3’-->5’ RNase activity of the enzyme. [J]. RNA (New York, N.Y.), 2008, 14(3).

[0007] [2] Dean F B, Nelson J R, Giesler T L, etc. Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification[J]. Cold Spring Harbor Laboratory Press, 2001, 11(6).

[0008] [3] Lian Dujuan, Qiu Jianping, Zhang Pingjing, etc. Research Progress on the Latest Applications of Phi29 DNA Polymerase[J]. Pharmaceutical Biotechnology, 2016, 23(02): 150–154.

[0009] [4] Torres L L, Pinheiro V B. Xenobiotic Nucleic Acid (XNA) Synthesis by Phi29 DNA Polymerase[J]. Current Protocols in Chemical Biology, 2018, 10(2): e41.

[0010] [5] Ja¨ger, S., and Famulok, M. (2004) Generation and enzymatic amplification of high-density functionalized DNA double strands. Angew. Chem., Int. Ed. 43, 3337–3340.

[0011] [6] Wong, K.K., and McClelland, M. (1991) PCR with 5-methyldCTP replacing dCTP. Nucleic Acids Res. 19, 1081–1085.

[0012] [7]Bailly,C.,and Waring,M.J.(1995)Transferring the purine 2-aminogroup from guanines to adenines in DNA changes the sequence-specific bindingof antibiotics.Nucleic Acids Res.23,885–892. Summary of the Invention

[0013] In order to overcome the disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide a method for preparing a hydrogel based on the activity of phi29 DNA polymerase rolling circle replication of long-chain base-modified nucleic acids.

[0014] Another object of the present invention is to provide an unnatural nucleic acid hydrogel prepared by the above method.

[0015] Still another object of the present invention is to provide the application of the above unnatural nucleic acid hydrogel.

[0016] The object of the present invention is achieved by the following technical solutions:

[0017] A method for preparing a hydrogel based on the activity of phi29 DNA polymerase rolling circle replication of long-chain base-modified nucleic acids, comprising the following steps:

[0018] (1) Preparation of nucleic acid with an amino linker on the base: Incorporate a pairable artificial nucleotide modified with propargylamino at the 5th position of the base part into the rolling circle amplification reaction system containing phi29 DNA polymerase to obtain nucleic acid with an amino linker on some bases.

[0019] (2) Preparation of unnatural nucleic acid hydrogel: Concentrate and anneal the nucleic acid obtained in step (1), mix the annealed product with a chemical crosslinking agent in phosphate buffer, and incubate to obtain the hydrogel.

[0020] The incorporation method in step (1) can be complete substitution or partial substitution of one or more of the natural nucleotides, and it is preferred that the content of the nucleic acid with an amino linker on the base in the finally obtained nucleic acid fragment is 10% - 40%.

[0021] The nucleic acid with an amino linker on the base in the nucleic acid fragment in step (1) is preferably distributed at intervals, more preferably evenly distributed at intervals, and the length is preferably 1000 - 5000 bp; more preferably 2000 bp.

[0022] The specific operations of step (1) are preferably as follows: Add the template and primers to the phi29 DNA polymerase buffer, and anneal and bind them; after annealing, add dNTPs, BSA (bovine serum albumin), and phi29 DNA polymerase, and incubate. The concentrations of each substance in the phi29 DNA polymerase buffer are calculated based on 1×phi29 DNA polymerase buffer containing: 5 - 20 nmol / L of template, 5 - 20 μmol / L of primers, 0.4 - 0.6 mmol / L of each dNTPs, 0.02 - 0.03 mg / mL of BSA, and 0.2 - 0.3 U / μL of phi29 DNA polymerase; more preferably, 10 nmol / L of template, 10 μmol / L of primers, 0.5 mmol / L of each dNTPs, 0.025 mg / mL of BSA, and 0.25 U / μL of phi29 DNA polymerase.

[0023] The conditions for the annealing and binding are 95 °C for 5 min, gradually cooled to room temperature (20 - 30 °C), and then placed on ice for 5 min; the conditions for the incubation are 27 - 32 °C for 10 - 14 h, more preferably 30 °C for 12 h.

[0024] The rolling circle amplification reaction described in step (1) is preferably carried out for 2 - 4 rounds.

[0025] The chemical cross - linker described in step (2) is preferably sulfosuccinimidyl ester poly(ethylene glycol) NHS - PEG n - NHS, where n in the PEG n is the number of ethylene glycol monomers in polyethylene glycol, and the value range of n is 2 - 12, more preferably 6.

[0026] The concentration in step (2) is preferably achieved by centrifuging at 6000 rpm for 30 min through a 30 kDa filter membrane.

[0027] The conditions for annealing in step (2) are preferably 95 °C for 5 min, gradually cooled to room temperature.

[0028] The final concentrations of each substance in the incubation system described in step (2) are preferably: 2 - 4 μg / μL of the annealed product, 2 - 4 mmol / L of the chemical cross - linker; the incubation conditions are 35 - 40 °C for 10 - 14 h; more preferably, 3 μg / μL of the annealed product, 3 mmol / L of the chemical cross - linker; the incubation conditions are 37 °C for 12 h.

[0029] The phosphate buffer described in step (2) is preferably a phosphate buffer with a pH of 8.4 - 8.6.

[0030] A non - natural nucleic acid hydrogel is prepared by the above - mentioned method.

[0031] Application of the above-mentioned unnatural nucleic acid hydrogel in the biomedical field.

[0032] The present invention has the following advantages and effects compared with the prior art:

[0033] The present invention explores the recognition of phi29 DNA polymerase for nucleotides with modifications on bases, expands the substrate spectrum of phi29 DNA polymerase, and on this basis, proposes a method for efficiently preparing nucleic acid hydrogels using an unnatural nucleic acid backbone. The nucleic acid with base modifications is amplified by the phi29 DNA polymerase rolling circle replication method to prepare a covalently cross-linked DNA hydrogel. There has never been a related study on preparing covalently cross-linked hydrogels by phi29 DNA polymerase rolling circle amplification before.

[0034] The present invention explores the recognition ability of phi29 DNA polymerase for nucleotides containing amino linkers and for unnatural base pairs. By enzymatically synthesizing nucleic acids with modifications on bases, covalent binding sites are provided. Since covalent bonds are more stable than hydrogen bonds, the stability of the DNA hydrogel can be improved. A large amount of nucleic acids can be quickly obtained through rolling circle amplification, providing a simple and highly universal production method for the formation of novel nucleic acid hydrogels. Description of the Drawings

[0035] Figure 1 It is a schematic diagram of the production process of the unnatural nucleic acid hydrogel of the present invention; among them, a) is the molecular structure of dCTP; b) is the molecular structure of 5-propargylamino-dCTP; c) is the structure of NHS-PEG6-NHS; d) is a schematic diagram of the preparation of a hydrogel by cross-linking unnatural nucleic acid and PEG.

[0036] Figure 2 It is a result diagram of verifying the recognition activity of phi29 DNA polymerase for base-modified nucleoside triphosphates (taking 5-propargylamino-dCTP as an example) by primer extension; among them, lane P0 is the primer; lane P1 is the positive control group, the extension product with natural dNTPs as the substrate; lane P2 is the experimental group, the extension product with 5-propargylamino-dCTP, natural dGTP, dTTP, and dATP as the substrate; lane P3 is the negative control group, the extension product (P3) with dNTPs without 5-propargylamino-dCTP (only natural dGTP, dTTP, and dATP) as the substrate.

[0037] Figure 3It is the electrophoretogram of the amplification product incorporated with base-modified nucleotide (5-propargylamino-dCTP) by phi29 DNA polymerase RCA; among them, lane 1 is the experimental group, with the RCA product using natural dGTP, dTTP, dATP and 5-propargylamino-dCTP containing modification; lane 2 is the control group, with the RCA product using natural dNTPs as the substrate.

[0038] Figure 4 It is the hydrogel morphology diagram; among them, a) is the diagram of rolling circle amplification for 3 rounds of nucleotide (5-propargylamino-dCTP) containing an amino linker; b) is the photo of the reaction product of the rolling circle amplification product for 3 rounds and NHS-PEG6-NHS.

[0039] Figure 5 It is the diagram of the observation results of the non-natural nucleic acid hydrogel by fluorescence microscopy and scanning electron microscopy; among them, a) is the fluorescence microscopy; b) is the scanning electron microscopy diagram. Specific implementation manners

[0040] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0041] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the technical field to which the present invention pertains.

[0042] The materials or reagents involved in the following embodiments:

[0043] The phi29 DNA polymerase is purchased from: New England Biolabs, model number M0269S;

[0044] 5-propargylamino-dCTP is purchased from: Wuhu Huaren Technology Co., Ltd., model number HR-00104022;

[0045] NHS-PEG6-NHS is purchased from: Shanghai Aladdin Biochemical Technology Co., Ltd., model number N164063;

[0046] The dNTP set is purchased from: New England Biolabs, model number N0446S;

[0047] The sequences involved in the following embodiments are shown in Table 1 below and are commissioned to be synthesized by Shanghai Bioengineering Co., Ltd.:

[0048] Table 1 Sequence names and sequences

[0049]

[0050] Note: N represents any base

[0051] Example 1 explores the recognition performance of phi29 DNA polymerase for base-modified nucleotide (5-propargylamino-dCTP)

[0052] 1) Primer extension using base-modified nucleoside triphosphate (5-propargylamino-dCTP):

[0053] Anneal the primer FAM-T75-R (1 μmol / L) modified with 5'-carboxyfluorescein (FAM) to the DNA template T1 (2 μmol / L) in 2×phi29 DNA polymerase buffer (reaction conditions: 95 °C, 5 min, gradually cool to room temperature and then place on ice for 5 min). Incubate the annealed template / primer mixture (0.5 μmol / L), natural dGTP, dTTP, dATP, and 0.4 mmol / L of modified 5-propargylamino-dCTP, BSA (0.05 mg / mL), and phi29 DNA polymerase (0.25 U / μL) in 1×phi29 DNA polymerase buffer at 30 °C for 12 h. In addition to the experimental group (P2), set up a positive control group: the extension product (P1) with natural dNTPs as the substrate; a negative control group: the extension product (P3) with dNTPs without 5-propargylamino-dCTP (only natural dGTP, dTTP, and dATP) as the substrate. Use 15% urea gel electrophoresis, and the extension bands containing the 5'-FAM primer can be directly presented through a gel imager. The results are as Figure 2 shown. In the experimental group, 5-propargylamino-dCTP was used to replace natural dCTP, and the band position of the extension product (P2) was basically the same as that of the positive control group (P1), while the negative control group (P3) could not extend without adding 5-propargylamino-dCTP. This indicates that phi29 DNA polymerase can recognize 5-propargylamino-dCTP and can synthesize nucleic acid chains containing propargylamino groups.

[0054] 2) Incorporation of base-modified (5-propargylamino-dCTP) nucleotides through phi29 DNA polymerase RCA:

[0055] In 1×phi29 DNA polymerase buffer, anneal the pUC19 plasmid as a template (10 nmol / L) with the hexamer random primer Hexamer (5 μmol / L) (reaction conditions: 95 °C, 5 min, gradually cool to room temperature and place on ice for 5 min). After annealing, add dNTPs (0.5 mmol / L), BSA (0.025 mg / mL), and phi29 DNA polymerase (0.25 U / μL). The control group uses natural dNTPs as the substrate, and the experimental group uses natural dGTP, dTTP, dATP, and modified 5-propargylamino-dCTP as the substrate. Incubate at 30 °C for 12 h in 1×phi29 DNA polymerase buffer. After the RCA reaction, use 1% agarose gel electrophoresis to verify whether there are macromolecules blocked in the wells, so as to verify whether long-chain DNA is formed. As Figure 3 , lane 1 is the RCA product with 5-propargylamino-dCTP replacing natural dCTP, and lane 2 is the RCA product with natural dNTPs as the substrate. Compared with the control group, the RCA product of the experimental group is slightly less, but there are obvious bands in the gel wells, indicating that phi29 DNA polymerase can integrate 5-propargylamino-dCTP into the nucleic acid chain through RCA to synthesize macromolecular long-chain DNA containing propargylamino.

[0056] Example 2 Preparation of unnatural nucleic acid hydrogel

[0057] The preparation method of the unnatural nucleic acid hydrogel is divided into two steps:

[0058] 1) Obtain long-chain nucleic acids containing propargylamino through 3 rounds of RCA. Add the hexamer random primer Hexamer (10 μmol / L) to 1×phi29 DNA polymerase buffer and anneal it to the plasmid pUC19 (10 nmol / L) (reaction conditions: 95 °C, 5 min, gradually cool to room temperature and place on ice for 5 min). After annealing, add dNTPs (0.5 mmol / L each of dGTP, dTTP, dATP, and 5-propargylamino-dCTP), BSA (0.025 mg / mL), and phi29 DNA polymerase (0.25 U / μL), and incubate at 30 °C for 12 h. After the first round of RCA, add the hexamer random primer Hexamer (5 μmol / L) to the system, anneal again, and add dNTPs (0.5 mmol / L each of dGTP, dTTP, dATP, and 5-propargylamino-dCTP), BSA (0.025 mg / mL), and phi29 DNA polymerase (0.25 U / μL), and incubate at 30 °C for 12 h. After the second round of reaction, repeat the second round of RCA operation again. The reaction product is asFigure 4 As shown in a), RCA can integrate long-chain nucleic acids containing propargylamino groups, but cannot directly form macromolecular crosslinks visible to the naked eye.

[0059] 2) The RCA product reacts with NHS-PEG6-NHS to form a hydrogel. After obtaining long-chain nucleic acids through three rounds of RCA, the amplified product is centrifuged at 6000 rpm for 30 min using a 30 kDa Amicon filter. The concentrated product is heated at 95 °C for 5 min and then gradually cooled to room temperature. The concentrated and annealed product (3 μg / μL) is reacted with NHS-PEG6-NHS (3 mmol / L) in 1×PBS (pH 8.5) buffer and incubated at 37 °C for 12 h to form a hydrogel. As Figure 4 shown in b), a hydrogel visible to the naked eye can be formed after the RCA product reacts with NHS-PEG6-NHS.

[0060] In summary, the RCA product cannot form a product visible to the naked eye, but a hydrogel visible to the naked eye can be formed after adding NHS-PEG6-NHS.

[0061] Example 3 Characterization of unnatural nucleic acid hydrogels

[0062] 1) Characterize the morphology using a fluorescence microscope: Take 10 μL of the macromolecular flocculent liquid formed by nucleic acid crosslinking, drop it on a glass slide, take 1 μL of Cyber Gold nucleic acid dye and mix it evenly, let it stand for 15 min, cover it with a coverslip, and adjust the fluorescence microscope to the green light channel for observation and photography. As Figure 5 shown in a), the hydrogel presents pores of different sizes.

[0063] 2) Characterize the morphology using a scanning electron microscope: Take 5 μL of the macromolecular flocculent liquid formed by nucleic acid crosslinking, drop it on a glass slide, and dry it using a freeze-drying vacuum dryer. Spray gold on the surface of the dried sample, and then it can be observed and photographed using a scanning electron microscope. Observe its internal fine structure, Figure 5 as shown in b), the hydrogel as a whole is lamellar.

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention. <110> South China University of Technology <120> Method for preparing hydrogel based on activity of phi29 DNA polymerase rolling circle replication of long-chain base-modified nucleic acid <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> T1 <400> 1 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc 60 ataaagtgta aagcc 75 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> FAM-T1-R <220> <223> 5'-end carboxyfluorescein (FAM) modification <400> 2 tggctttaca ctttatgctt ccg 23

Claims

1. A method for preparing a hydrogel based on the activity of phi29 DNA polymerase rolling circle replication of long-chain base-modified nucleic acids, characterized in that: It includes the following steps: (1) Preparation of nucleic acid with an amino linker on the base: Add the template and primer to the phi29 DNA polymerase buffer and anneal them for binding; after annealing, add dNTPs, bovine serum albumin, and phi29 DNA polymerase. In the dNTPs, a pairable artificial nucleotide 5-propargylamino-dCTP with an alkyne-propylamino modification at the 5th position of the base part is incorporated to obtain a rolling circle amplification reaction system of phi29 DNA polymerase; incubate at 27 - 32 °C for 10 - 14 h to prepare nucleic acid with an amino linker on some bases. (2) Preparation of unnatural nucleic acid hydrogel: Concentrate and anneal the nucleic acid obtained in step (1), mix the annealed product with a chemical crosslinker in phosphate buffer, and incubate to obtain the hydrogel. The chemical crosslinking agent is sulfosuccinimidyl ester polyethyleneglycol NHS-PEG n -NHS, where PEG n in which n is the number of ethylene glycol monomers in polyethylene glycol, and the value of n is 2 to 12; The final concentrations of each substance in the incubation system are as follows: annealed product 2 - 4 μg / μL, chemical crosslinker 2 - 4 mmol / L; the incubation conditions are 35 - 40 °C for 10 - 14 h.

2. The method for preparing a hydrogel based on the activity of rolling circle replication of long-chain base-modified nucleic acid by phi29 DNA polymerase according to claim 1, wherein: The value of n is 6.

3. The method for preparing a hydrogel based on the activity of rolling circle replication of long-chain base-modified nucleic acid by phi29 DNA polymerase according to claim 1 or 2, wherein: In step (2), the concentration is achieved by centrifuging through a 30 kDa filtration membrane at 6000 rpm for 30 min; In step (2), the annealing conditions are 95 °C for 5 min, and gradually cooled to room temperature.

4. The method for preparing a hydrogel based on the activity of rolling circle replication of long-chain base-modified nucleic acid by phi29 DNA polymerase according to claim 1 or 2, wherein: The phosphate buffer in step (2) is a phosphate buffer with a pH of 8.4 - 8.

6.

5. The method for preparing a hydrogel based on the activity of rolling circle replication of long-chain base-modified nucleic acid by phi29 DNA polymerase according to claim 1 or 2, wherein: In step (1), the incorporation method is to completely or partially replace one or more of the natural nucleotides, and the content of nucleic acid with an amino linker on the base in the finally obtained nucleic acid fragment is 10% - 40%; In step (1), the nucleic acid with an amino linker on the base in the nucleic acid fragment is distributed at intervals and has a length of 1000 - 5000 bp.

6. The method for preparing a hydrogel based on the activity of rolling circle replication of long-chain base-modified nucleic acid by phi29 DNA polymerase according to claim 1, wherein: The concentrations of each substance in the rolling circle amplification reaction system of phi29 DNA polymerase in step (1) are as follows: template 5 - 20 nmol / L, primer 5 - 20 μmol / L, each dNTP 0.4 - 0.6 mmol / L, bovine serum albumin 0.02 - 0.03 mg / mL, phi29 DNA polymerase 0.2 - 0.3 U / μL.

7. The method for preparing a hydrogel based on the activity of phi29 DNA polymerase rolling circle replication of long-chain base-modified nucleic acids according to claim 1, characterized in that: The annealing and binding conditions in step (1) are 95 °C for 5 min, gradually cooled to room temperature and then placed on ice for 5 min.

8. A non-natural nucleic acid hydrogel, characterized in that: Prepared by the method according to any one of claims 1 to 7.

9. Use of the unnatural nucleic acid hydrogel according to claim 8 in the preparation of a drug carrier.

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