CN114790471A

CN114790471A - Non-natural nucleic acid hydrogel and preparation method and application thereof

Info

Publication number: CN114790471A
Application number: CN202110101329.7A
Authority: CN
Inventors: 陈庭坚; 张汝洁; 彭乐丽; 叶方开; 雷谨铭; 何传平; 王光远
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2022-07-26
Anticipated expiration: 2041-01-26
Also published as: CN114790471B

Abstract

The invention discloses a non-natural nucleic acid hydrogel and a preparation method and application thereof, belonging to the technical field of biological materials. The method of the invention operates as follows: the method comprises the steps of doping artificial nucleotides which can be matched and contain propargylamine group modification at the 5 th position of a basic group part into a polymerase chain reaction system, preparing a nucleic acid fragment containing an amino connecting arm on a part of basic groups, reacting with a click chemical joint, carrying out click chemical reaction with primers containing corresponding groups at the 5' end to obtain a ' hairbrush ' primer, carrying out PCR amplification by using the nucleic acid fragment or plasmid containing a target fragment as a template, concentrating an amplification product, and carrying out high-temperature annealing. The method provided by the invention is simple and easy, has high universality, and the obtained hydrogel can protect functional DNA so that the functional DNA is not easy to degrade in serum, thereby not only overcoming the defects of the traditional DNA hydrogel, but also having the advantages of higher stability, controllable crosslinking degree, carrying genetic information and the like, and having good application prospect.

Description

Non-natural nucleic acid hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a non-natural nucleic acid hydrogel and a preparation method and application thereof.

Background

The hydrogel is a common material, wherein part of the hydrogel has good biocompatibility, adjustable biodegradability and controllable mechanical properties, and has important application value in the fields of chemical and biomedical engineering. With the research in the hydrogel field, more precise synthesis methods, more sensitive stimuli responsiveness and more abundant functionalities have become the main research direction and target in the development process of novel application type hydrogels.

DNA is the core genetic material of the living system, directing the development and functioning of vital functions of the organism. From the material chemistry perspective, DNA is a natural biopolymer with incomparable characteristics with synthetic polymers. For example: the base complementary pairing property enables the DNA to have accurate and efficient self-assembly capability; the DNA sequence is various and adjustable, the structure is accurate and controllable, and the stimulation responsiveness is rich; natural evolution endows organisms with abundant and various biological enzymes, and DNA can be accurately operated at a molecular level; the DNA has good biocompatibility and biodegradability.

In recent years, scientists have used DNA as a building element to synthesize a variety of DNA functional polymer materials. Among them, DNA hydrogel is an important DNA material, and is a three-dimensional polymer network constructed by using DNA as a structural motif, and has been developed vigorously in recent years. The DNA hydrogel not only utilizes the skeleton structure of the hydrogel, but also retains the biological function of the DNA, realizes the perfect fusion of the structure and the function of the hydrogel material, and has wide application prospect in the fields of biosensors, drug delivery, cell culture, protein synthesis, intelligent devices, environmental protection and the like.

Several methods for preparing DNA hydrogels have been reported. Preformed DNA building blocks (X, Y, T-type DNA) are interacted with complementary DNA strands, for example, by control of enzymes or pH. DNA hydrogels can also be prepared by PCR amplification or multi-primer rolling circle replication using Y-shaped DNA primers. However, these hydrogels are formed by hydrogen bonding between bases, and have low stability, special sequence design, and limited controllability of many properties (such as density, mechanical properties, etc.), which greatly limits the popularization and application of DNA hydrogels.

Disclosure of Invention

In order to overcome the disadvantages and shortcomings of the prior art, the present invention provides a method for preparing a non-natural nucleic acid hydrogel, which is a method for efficiently preparing a nucleic acid hydrogel having more excellent properties by using a non-natural nucleic acid backbone.

Another object of the present invention is to provide a non-natural nucleic acid hydrogel obtained by the above-mentioned production method.

It is still another object of the present invention to provide the use of the above-mentioned non-natural nucleic acid hydrogel.

The purpose of the invention is realized by the following technical scheme:

a method for preparing a non-natural nucleic acid hydrogel, comprising the following steps:

(1) preparation of upstream and downstream 'hairbrush' primers:

a. the method comprises the following steps of (1) doping mateable 5-site propargylamine-containing modified artificial nucleotide of a base part into a polymerase chain reaction system to prepare a nucleic acid fragment containing an amino connecting arm on a part of bases;

b. b, reacting the nucleic acid fragment obtained in the step a with a click chemical linker, and modifying the linker on partial nucleotides of the nucleic acid fragment;

c. designing and synthesizing an upstream primer and a downstream primer according to the sequence of the target fragment, and modifying groups which can generate click chemical reaction with the joint at the 5' ends of the upstream primer and the downstream primer;

d. c, respectively carrying out click chemical reaction on the nucleic acid fragment containing the joint on part of the nucleotides obtained in the step b and the upstream and downstream primers containing the modification group at the 5' end obtained in the step c to obtain upstream and downstream hairbrush primers;

(2) preparation of non-natural nucleic acid hydrogel:

and (2) taking a nucleic acid segment or plasmid containing a target segment as a template, carrying out PCR amplification by using the upstream and downstream hairbrush primers obtained in the step (1), concentrating an amplification product, and carrying out high-temperature annealing to obtain the non-natural nucleic acid hydrogel.

The polymerase in the polymerase chain reaction system in the step a can be any one of Taq polymerase, OneTaq polymerase, Q5 polymerase or SFM4-3 polymerase;

when the polymerase is SFM4-3, the artificial nucleotide which can be paired and contains the modification of the fluorine-containing group on the No. 2 site of the glycosyl can be added into a polymerase chain reaction system to prepare the nucleic acid fragment containing the amino modification on the basic group and the fluorine-containing modification on the glycosyl, and then the subsequent reaction is carried out.

When the polymerase is SFM4-3, the non-natural bases dTPT3 and dNaM can be added into a polymerase chain reaction system to prepare a nucleic acid fragment containing non-natural base pairs, and then the subsequent reaction is carried out.

The nucleotide in the polymerase chain reaction system can be a deoxyribonucleotide mixture, a ribonucleotide mixture or a deoxyribonucleotide/ribonucleotide mixture, and corresponding nucleic acid fragments are prepared and then subjected to subsequent reaction.

The incorporation mode in step a can be complete or partial substitution of one or more of natural nucleotides, preferably 10% to 40% of the nucleic acid content containing the amino linker arm on the base in the finally obtained nucleic acid fragment.

The nucleic acid containing the amino connecting arms on the bases in the nucleic acid fragment in the step a is preferably distributed at intervals, more preferably distributed at equal intervals, and the length of the nucleic acid is preferably 50-200 bp; more preferably 70-100 bp; most preferably 75 bp.

The click chemistry linker in step b may be selected from the group consisting of NHS-DBCO (diphenylcyclooctyne-succinimidyl ester) linker, NHS-TCO (Trans-cyclooctene-succinimidyl ester) linker; preferably a NHS-DBCO linker.

The optimal proportion of the nucleic acid fragment to the click chemical joint in the step b is 1: 1000-1500 molar ratio; more preferably 1: 1000.

The reaction conditions in the step b are preferably 35-40 ℃ and 10-15 h, and more preferably 37 ℃ and 12 h.

The reaction in step b is preferably carried out in a PBS buffer system at pH 8.4 to 8.6.

The optimal molar ratio of the nucleic acid fragment to the upstream primer or the downstream primer in the step d is 8-12: 1; more preferably 10: 1.

The reaction system in the step d also contains NaCl and Tween 20; more preferably, the composition is as follows: 0.8-1.2 mol/L NaCl, 0.04-0.06% Tween20, 2-4 mu mol/L nucleic acid fragment, and 20-40 mu mol/L5-end upstream primer or downstream primer; most preferably the composition is as follows: 1mol/L NaCl, 0.05% Tween20, 3.3. mu. mol/L nucleic acid fragment, 33. mu. mol/L5-terminal upstream primer or downstream primer.

The group capable of reacting with the linker in step c is preferably an azide group.

The reaction conditions in the step d are preferably 45-55 ℃ and 10-15 h, and more preferably 50 ℃ and 12 h.

The reaction in step d is preferably carried out in a PBS buffer system with a pH of 7.3 to 7.5.

The conditions for the concentration in step (2) are preferably as follows: centrifuging at the rotating speed of 5000-7000 rpm for 25-35 min; more preferably as follows: centrifuging at 6000rpm for 30 min.

The operation of the high-temperature annealing described in the step (2) is preferably as follows: heating at 95 deg.C for 5min, and gradually cooling to room temperature.

The target fragment in step c and step (2) may be a gene fragment or other non-gene fragment with a specific sequence. The sequence of the target fragment depends on the purpose and application, and the length is preferably 0-3000 bp, more preferably 100-1500 bp. Wherein, the gene can be all genes which can be used for transcribing RNA aptamer or expressing protein, such as Broccoli, GFP, beta lactamase gene and the like.

A non-natural nucleic acid hydrogel prepared by the above method.

The application of the non-natural nucleic acid hydrogel in the biomedical field.

The invention synthesizes nucleic acid with modified base through enzyme method, provides covalent binding site, and solves the problem of low stability of DNA hydrogel through introducing covalent bond. The nucleotide with modified base is cheap and easy to obtain, can be recognized by various nucleic acid polymerases, and provides a convenient preparation method for forming a hydrogel nucleic acid skeleton. Meanwhile, by controlling the sequence of the nucleic acid skeleton, the properties of the formed hydrogel, such as density degree, mechanical property, stability and the like, can be conveniently controlled and optimized. On the basis, the invention also uses the polymerase-Taq DNA polymerase Stoffel fragment mutant strain SFM4-3 subjected to directed evolution, simultaneously integrates nucleotides with modifications on the base and the glycosyl group and introduces unnatural base pairs (dTPT3/dNaM) through PCR, provides more connection sites for the introduction of covalent bonds, and thus increases the potential application of the hydrogel (for example, the modification on the base introduces the covalent bonds for forming the hydrogel, and the modification on the glycosyl group introduces the covalent bonds for connecting the drug molecules capable of being slowly released). Meanwhile, polymerase SFM4-3 can synthesize a hybrid nucleic acid chain of deoxyribonucleoside and ribonucleotide, and provides a new method for forming hybrid hydrogel by using deoxynucleotide and non-deoxynucleotide.

Compared with the prior art, the invention has the following advantages and effects:

the invention researches a simple and easy preparation method of nucleic acid hydrogel with high universality. The hydrogel can protect functional DNA from degradation in serum, and can carry, transcribe and translate genetic information. The hydrogel prepared by the method overcomes the defects of the traditional DNA hydrogel, has the advantages of high stability, controllable crosslinking degree, genetic information carrying and the like, and increases the potential application of the DNA hydrogel on the basis. The nucleic acid hydrogel prepared by using the nucleic acid modified by the basic group overcomes the following defects of the traditional nucleic acid hydrogel:

1. the stability is low;

2. the preparation materials are not easy to obtain, and the universality of the preparation method is low;

3. controllability of properties (e.g. degree of density, mechanical properties, etc.) is limited.

Increase the potential applications of DNA hydrogels:

1. provides a plurality of covalent bond introduction sites, so that the nucleic acid hydrogel is easy to carry functional substances;

2. the method can be used for preparing nucleotide and deoxynucleotide heterozygous hydrogel, and widens the application scene of nucleic acid hydrogel;

3. can be used for wrapping functional DNA, so that the functional DNA is not easily degraded in serum;

4. can be used for carrying, transcribing and translating genetic information;

5. the sites of covalent bonding can be precisely controlled, and the degree of crosslinking of the hydrogel can be controlled.

Drawings

FIG. 1 is a schematic diagram of a process for preparing the non-natural nucleic acid hydrogel of the present invention.

FIG. 2 is a diagram showing the results of electrophoresis of PCR amplification products of different hairbrush primers in example 1; wherein, figure A: lane M is Marker, lane 1 is the common primer PCR results, lane 2 is the Broccoli "brush primer" PCR results, Panel B: lane M is Marker, and lane 1 is the GFP "Brush primer" PCR result.

FIG. 3 is a photograph of a Broccoli gene-containing non-native nucleic acid hydrogel prepared in example 1; wherein, the picture A is a picture of the micelle before annealing, and the picture B is a picture of the hydrogel under ultraviolet light after annealing at high temperature and staining by Cyber Gold nucleic acid gel electrophoresis dye.

FIG. 4 is a graph showing the result of fluorescent microscope observation of the Broccoli gene-containing non-natural nucleic acid hydrogel prepared in example 1 after annealing and concentration.

FIG. 5 is a graph showing the result of scanning electron microscope observation of the non-native nucleic acid hydrogel containing a Broccoli gene prepared in example 1.

FIG. 6 is a diagram showing the real-time detection of the transcript of the hairbrush PCR product containing the Broccoli gene by the microplate reader in example 2.

FIG. 7 is a graph showing the expression of green fluorescent protein in a cell-free system by the hairbrush PCR product containing the GFP gene of example 2; wherein, 1 is a negative control group without a template; 2 is a hairbrush PCR negative control product group; 3 is a linear template positive control group; 4 is an azide modified template positive control group; 5 hairbrush PCR product group I (15 ng/. mu.l); 6 is hairbrush PCR product test group II (45 ng/. mu.l); 7 is hairbrush PCR product run III (75 ng/. mu.l).

FIG. 8 is a graph showing the comparison of fluorescence intensity of hairbrush PCR products containing GFP gene in example 2 with that of isoconcentration linear DNA after protein expression in a cell-free system.

FIG. 9 is a graph showing the degradation of hairbrush PCR products containing Broccoli gene in fetal calf serum in example 2.

FIG. 10 is a graph showing the transcription of the brush PCR product non-native nucleic acid hydrogel containing the Broccoli gene in example 2 after 4 hours of degradation in fetal bovine serum.

FIG. 11 is a graph showing the results of testing the ability of extension of dNTPs/NTPs; wherein, panel A is dNTPs and panel B is NTPs.

FIG. 12 is a photograph of electrophoresis of base-modified (5-propargylamino-dCTP) nucleotides introduced by PCR using different polymerases; wherein, the picture A is SFM4-3 polymerase PCR product, and the picture B is Q5, OneTaq and Taq polymerase PCR product.

FIG. 13 is a nucleotide electrophoresis diagram of simultaneous incorporation of a base modification (5-propargylamino-dCTP) and a sugar modification (2' -fluoro-UTP) by SFM4-3 polymerase PCR.

FIG. 14 shows an electrophoretogram of simultaneous incorporation of base-modified (5-partylamino-dCTP) nucleotides and unnatural base pairs (dTPT3/dNaM) by SFM4-3 polymerase PCR; wherein, panel A is PCR product with base modification and non-natural base pair (p: PCR product, S1: conjugate of dTPT3-biotin with streptavidin on PCR product, S2: reactant of modified amino and NHS-biotin on PCR product, t: S1 and S2 with streptavidin conjugate), panel B incorporates both sugar-based modified (2' -fluoro-CTP) nucleotide and non-natural base pair (dTPT3/dNaM) electrophoretogram by SFM4-3 polymerase PCR (p: PCR product, S: dPT 3-biotin with streptavidin conjugate on PCR product).

FIG. 15 is an electrophoretogram of simultaneous incorporation of ribose and deoxyribose nucleotides by SFM4-3 polymerase PCR.

FIG. 16 is a graph showing the reaction results of different densities of amino-modified PCR products with NHS-FAM in example 4; wherein lane 1 shows a DNA product band of 5-propargylamino-dCTP separated by 5 bases, and lane 2 shows a DNA product band of 5-propargylamino-dCTP separated by 10 bases.

FIG. 17 is a graph showing the results of reaction of DNA prepared in example 1, in which bases have amino group modifications, using different linkers in example 4; wherein, the graph A is a graph of the reaction result of different linkers and DNA containing modified bases, and the graph B is a graph of the reaction of nucleic acid connected with NHS-DBCO and primer modified by 5' terminal azide.

FIG. 18 is a graph showing the results of sequencing of 9 samples obtained in example 5.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

Materials or reagents referred to in the following examples:

SFM4-3 polymerase, SF-WT polymerase: it is described in the literature "Chen T, Hongdilokul N, Liu Z, et al.

Evolution of thermophilic DNA polymerases for the recognition and amplification of

C2-modified DNA [ J ] Nature Chemistry,2016.

Deep Vent polymerase was purchased from: new England Biolabs model M0258S

Q5 polymerase was purchased from: new England Biolabs model M0491S

OneTaq polymerase was purchased from: new England Biolabs model M0480L

Taq polymerase was purchased from: new England Biolabs model M0267S

5-procargylamino-dCTP was purchased from: model HR-00104022 from Wenhu Huaren science Co Ltd

2' -fluoro-CTP was purchased from: model HR-00104018 from Wen u Huaren science and technology Co Ltd

2' -fluoro-UTP was purchased from: model HR-00104020 from Wen u Huaren science and technology Co Ltd

2' -methoxy-GTP was purchased from: model HR-00104003 from Wen u Huaren science and technology Co Ltd

dNTP sets were purchased from: new England Biolabs model N0446S

The rNTP package was purchased from: new England Biolabs model N0450S

dPT 3-biotin, dNaM already described in the literature "Li L, Degardin M, Lavergne T, et al

Replication of an Unnatural Base Pair for the Expansion of the Genetic Alphabet and

Biotechnology Applications[J].Journal of the American Chemical Society,2014,

136(3) 826 and 829

Streptavidin was purchased from: new England Biolabs model N7021S

Fetal bovine serum was purchased from: ExCell Bio, model FSP500

NHS-DBCO was purchased from Guangzhou hundred million waves Biotechnology Inc. under model bcd6

NHS-TCO was purchased from: click Chemistry Tools, model 1016-25

DHFBI was purchased from: glpbio corporation, USA, model GC30098

NHS-FAM was purchased from: shanghai Tuo Yang Biotechnology Limited, model HY-15938

1 × SF polymerase buffer formulation: 50mmol/L Tris-HCl (pH 8.5), 50mmol/LKCl, 6.5mmol/LMgCl ₂

Cyber Gold purchased from: bai Fluor Biotech Ltd, model number TJ702

EcoRI was purchased from: saimei Feishell science & Tech, model FD0274

HindIII was purchased from: saimer Feishell science & ltd.FD 0504

T4 ligase was purchased from: new England Biolabs model M0202S

1 × binding and washing buffer formulation: 10mmol/L Tris-HCl (pH7.4), 1mol/L NaCl, 0.1% Tween20, 1mmol/L EDTA

Magnesium acetate was purchased from: hadamard reagent Inc. model 01023011

Calcium folinate hydrate was purchased from: hadamard reagent, Inc. model number 25231A

Sodium pyruvate was purchased from: hadamard reagent Inc. model 01009805

Ammonium hydroxide solution was purchased from: shanghai Michelin Biochemical technology Ltd, model A801005

Oxalic acid was purchased from: hadamard reagent, Inc. model number 01023951

HEPES was purchased from: hadamard reagent, Inc. model number 01110191

L-glutamic acid monopotassium salt monohydrate was purchased from: hadamard reagent, Inc. model 49601

1, 4-diaminobutane was purchased from: hadamard reagent Inc. model 01007891

Spermidine was purchased from: hadamard reagent, Inc. model S02660

Phospho (enol) pyruvate monopotassium salt (PEP) was purchased from: Sigma-Aldrich, model number P7127

Potassium hydroxide was purchased from: shanghai Maxin Biochemical technology Ltd, model No. P822103

L-alanine was purchased from: hadamard reagent, Inc. model number 01088967

L-arginine was purchased from: hadamard reagent, Inc. model number 01111537

L-asparagine was purchased from: hadamard reagent Inc. model 01107585

L-aspartic acid was purchased from: hadamard reagent Inc. model 01089459

L-cysteine was purchased from: hadamard reagent Inc. model 01083581

L-glutamic acid was purchased from: shanghai Shaoshao reagent GmbH, model SY008812

L-Glutamine was purchased from: hadamard reagent Inc. model 01089485

Glycine was purchased from: hadamard reagent, Inc. model number 01088948

L-histidine was purchased from: hadamard reagent Inc. model 01108001

L-isoleucine was purchased from: hadamard reagent Inc. model TZL36951

L-leucine was purchased from: hadamard reagent, Inc. model number 01096937

L- (+) -lysine was purchased from: hadamard reagent Inc. model 01089505

L-methionine was purchased from: hadamard reagent, Inc. model number 01100470

L-phenylalanine was purchased from: hadamard reagent Inc. model 01100850

L proline was purchased from: hadamard reagent, Inc. model number 01025290

L-serine was purchased from: hadamard reagent Inc. model 01089010

L-threonine was purchased from: hadamard reagent Inc. model 01109021

L tryptophan was purchased from: hadamard reagent Inc. model 01109787

L-tyrosine was purchased from: hadamard reagent Inc. model 01094035

L-Valine was purchased from: hadamard reagent Inc. model 01109011

The L-glutamic acid hemi magnesium salt tetrahydrate was purchased from: sigma, model 49605

Sodium oxalate was purchased from: sigma-aldrich, model 71800

Beta-nicotinamide adenine dinucleotide was purchased from: Sigma-Aldrich, model N6522

Adenosine triphosphate was purchased from: saimer Feishale science, Inc. model R0481

Guanosine triphosphate was purchased from: saimei Feishire science and technology, model R0481

Cytosine nucleoside triphosphates were purchased from: saimei Feishire science and technology, model R0481

Uridine triphosphates were purchased from: saimei Feishire science and technology, model R0481

tRNA (from E.coli MRE 600, Roche, model 10706640

Coenzyme a hydrates were purchased from: sigma-aldrich, model C4282

Dithiothreitol was purchased from: hadamard reagent Inc. model 01064273

Dipotassium hydrogen phosphate was purchased from: shanghai Tantake Technique GmbH, model V900050

Potassium dihydrogen phosphate was purchased from: shanghai Tantake Technology GmbH, model G82821B

Yeast extracts were purchased from: shanghai Tantake Tech Ltd, model LP0021

Protein was purchased from: shanghai Tantake Tech Ltd, model LP0042

Sodium chloride was purchased from: shanghai Tantake Technologies GmbH, model number G81793H

The sequences involved in the following examples are shown in Table 1 below, and were synthesized by Shanghai bioengineering, Inc.:

TABLE 1 sequence names and sequences

Example 1 preparation of non-Natural nucleic acid hydrogel

(1) Obtaining modified base-containing DNA:

a template T1(4nmol/L, 75bp, Table 1), an upstream primer T1-F (0.3. mu. mol/L, Table 1), a downstream primer T1-R (0.3. mu. mol/L, Table 1), and Q5 polymerase (20U/ml, each 0.2mmol/L of natural dGTP, dTTP, dATP, and 5-proparylamino-dCTP containing a modification) were added to 1 XQ 5 polymerase buffer to carry out PCR (reaction conditions: 98 ℃ for 2 min; 98 ℃ for 10s, 70 ℃ for 30s, and 72 ℃ for 1min, and the reaction was repeated 20 times; 72 ℃ for 5min) to obtain a PCR product containing 5-proparylamino-dCTP containing a modification in base, and the product was purified using a DNA purification kit.

(2) Making up upstream and downstream 'hairbrush primers':

mu.mol/L of the PCR product containing the modified 5-propargylamino-dCTP obtained in step (1) and 1mmol/L of NHS-DBCO were added to 1 XPBS (pH 8.5) buffer, and after incubation at 37 ℃ for 12 hours, the reaction was checked by electrophoresis on a 6% polyacrylamide gel. The reaction product was centrifuged 6 times for 15min each time with a 30kDa Amicon filter at 6000rpm in 1 XPBS (pH7.4).

1mol/L NaCl, 0.05% Tween20, 3.3 mu mol/L PCR product after reaction with NHS-DBCO, and upstream primer N with azide modification at 5 terminal are added into 1 XPBS (pH7.4) buffer solution ₃ -Broccoli-F (33. mu. mol/L, Table 1) or N ₃ GFP-F (40. mu. mol/L, Table 1), 5-Azide modified downstream primer N ₃ -Broccoli-R (33. mu. mol/L, Table 1) or N ₃ GFP-R (40. mu. mol/L, Table 1), incubated at 50 ℃ for 12h, and checked for reaction by gel electrophoresis on 6% PAGE gels. The reaction product was centrifuged 6 times for 15min at 6000rpm in 1 XPBS (pH7.4) using a 30kDa Amicon filter. The manufacturing process is shown in fig. 1.

(3) Construction of a plasmid containing the Broccoli Gene:

to 1 XThermopol reaction buffer was added template T2(4nmol/L, Table 1), upstream primer T2-F-P (0.5. mu. mol/L, Table 1) containing the start codon, downstream primer T2-R-T (0.5. mu. mol/L, Table 1) containing the stop codon, 0.2. mu. mol/L dNTPs, Taq polymerase 25U/mL, and PCR was carried out (reaction conditions: 95 ℃ 30 s; 95 ℃ 30s, 63 ℃ 30s, 68 ℃ 15s, 30 cycles; 68 ℃ 5 min). The Broccoli gene carrying the start and stop codes was obtained by PCR. And purifying the PCR product to be used as a template for the next PCR.

The PCR reaction was carried out by adding 37nmol/L, 0.2. mu. mol/L dNTP, the upstream primer T2-F-E (0.2. mu. mol/L, Table 1) containing the EcoRI cleavage site, the downstream primer T2-R-H (0.2. mu. mol/L, Table 1) containing the HindIII cleavage site, and 25U/mL Taq polymerase to 1 XThermopol reaction buffer (reaction conditions: 95 ℃ 30 s; 95 ℃ 30s, 65 ℃ 1 min; 68 ℃ 20s, 30 cycles; 68 ℃ 5 min). The method comprises the steps of carrying out enzyme digestion on a Broccoli gene fragment with an enzyme digestion site and a pUC57 plasmid, adding the Broccoli gene fragment and the pUC57 plasmid into a 1 XFastdigest Green buffer solution respectively, adding EcoRI and HindIII in proportion, reacting for 12 hours at 37 ℃, adding the fragment and the vector after enzyme digestion purification into a 1 XT 4 DNA ligase buffer solution in proportion of 3:1, using T4 DNA ligase to ligate the Broccoli gene and the pUC57 plasmid, ligating for 12 hours at 16 ℃, transferring a ligation product into a DH5 alpha competent cell, and obtaining a large amount of plasmids containing the Broccoli gene.

(4) Preparation of hydrogel and concentration:

preparation of hydrogel containing Broccoli gene: 1nmol/L of the above-mentioned Broccoli gene-containing template, 0.2mmol/L dNTP, 43nmol/L each of a Broccoli upstream "brush primer" and a Broccoli downstream "brush primer", 1mol/L NaCl, 0.05% Tween20, and 50U/mL OneTaq (94 ℃ 2 min; 94 ℃ 30s, 50 ℃ 1min, 68 ℃ 5min, 30 cycles; 68 ℃ 10min) were added to 1 XOneTaq buffer, respectively. Gel electrophoresis was performed using 1% agarose to verify the presence of large molecules trapped in the wells, and the results are shown in FIG. 2A (1 is a negative control: common primer PCR product, and 2 is brush primer PCR product).

Preparation of hydrogel containing GFP gene: 4nmol/L of GFP template gene containing T7 promoter and terminator (Table 1), 0.2mmol/L dNTP, 48nmol/L of each of GFP upstream "brush primer" and GFP downstream "brush primer", 1mol/L NaCl, 0.05% Tween20, 50U/mL OneTaq (94 ℃ 5 min; 94 ℃ 30s, 65 ℃ 1min, 68 ℃ 5min, cycle 35 times; 68 ℃ 10min) were added to 1 XOneTaq buffer. Gel electrophoresis using 1% agarose was performed to verify the presence of large molecules trapped in the wells, and the results are shown in FIG. 2B.

Taking hydrogel containing Broccoli gene, centrifuging with 30kDa Amicon filter at 6000rpm for 30min, heating at 95 deg.C for 5min, and gradually cooling to room temperature to form gel. As shown in FIG. 3A, the macromolecules formed by PCR are pooled and concentrated in the form of loose micelles; as shown in FIG. 3B, the hydrogel was denser after high-temperature annealing, and after staining with Cyber Gold, significant fluorescence was observed under ultraviolet light, thereby proving that the main component of the hydrogel was nucleic acid.

(6) Non-native nucleic acid hydrogel characterization:

when the hydrogel containing the Broccoli gene was observed by a fluorescence microscope, the fine mesh-like structure of the hydrogel was observed as shown in FIG. 4.

After freeze-drying the hydrogel and gold-plating the surface, the fine structure inside was observed by Scanning Electron Microscopy (SEM) and found to be in the shape of a nanoflower, as shown in FIG. 5.

EXAMPLE 2 use of non-Natural nucleic acid hydrogels

1. Transcribable the genetic information carried

Adding a hairbrush PCR product containing the Broccoli gene into a reaction system containing T7 transcriptase, adding a fluorescent molecule DFHBI capable of reacting with the transcribed RNA aptamer, and monitoring the fluorescence intensity in real time through an enzyme-labeling instrument so as to reflect the Broccoli transcription condition. The control group was a transcription system without hydrogel under the same conditions. The results are shown in FIG. 6. This result confirms that the hydrogel carrying the genetic information can be transcribed.

2. Can express the carried genetic information

Expressing a hairbrush PCR product containing a GFP gene in an escherichia coli cell-free system, and finally, determining the concentration of each reagent in a cell-free system reaction system: 1.2mmol/L adenosine triphosphate, 0.850mmol/L guanosine triphosphate, 0.850mmol/L uridine triphosphate, 0.850mmol/L cytosine triphosphate, 31.50. mu.g/mL folinic acid, 170.60. mu.g/mL tRNA, 0.40mmol/L Nicotinamide Adenine Dinucleotide (NAD), 1.50mmol/L spermidine and 57.33mmol/L HEPES buffer, 10mmol/L magnesium glutamate, 10mmol/L ammonium glutamate, 130mmol/L potassium glutamate, 2mmol/L each of 20 amino acids, 0.03mol phosphoenolpyruvate (PEP), 0.27mmol/L coenzyme A (CoA), 4.00mmol/L oxalic acid, 1.00mmol/L putrescine. The reaction conditions were water bath 30 ℃ overnight. Experimental groups were set as follows: 1: no template negative control group; 2: a hairbrush PCR negative control product group (hairbrush reaction system without PCR cross-linking); 3: wire(s)Positive control group for sexual template (with GFP-F, GFP-R (SEQ ID NO: N) without any modification ₃ -GFP-F、N ₃ GFP-R) is 40 ng/. mu.l of PCR purified product obtained by primer amplification); 4: azide-modified template positive control group (with N) ₃ -GFP-F、N ₃ GFP-R is a PCR purified product obtained by primer amplification, and the concentration is 40 ng/. mu.l); 5: hairbrush PCR product Experimental group I (hairbrush PCR product dialysate 15 ng/. mu.l); 6: hairbrush PCR product experiment group II (hairbrush PCR product dialysate 45 ng/. mu.l); 7: hairbrush PCR product run group III (hairbrush PCR product dialysate 75 ng/. mu.l). As shown in FIG. 7, the expression differences of the linear template, the azide-modified template and the hairbrush negative control product are compared, which shows that the hairbrush hydrogel can be used for in vitro transcription and translation and has better effect.

Two sets of control experiments were set and three parallel controls were made for each set, as shown in fig. 8, the cell-free protein expression system containing and not containing the hairbrush PCR product was reacted for 20 minutes at 37 ℃, and by monitoring the fluorescence intensity, it was verified again that the hairbrush hydrogel could be used for in vitro transcription and translation with better effect.

3. Can protect DNA carrying genetic information from being easily degraded in fetal calf serum

Respectively placing macromolecules obtained by hairbrush primer PCR with the same nucleic acid concentration (274 ng/. mu.l) and Broccoli DNA amplified by normal primer PCR in fetal calf serum, degrading for 12 hours, analyzing the fluorescence intensity of bands by gel electrophoresis of two degradation products every hour, and obtaining the change curve of the fluorescence intensity of the two bands. As shown in FIG. 9, it can be seen that under the same conditions, DNA is almost completely degraded within 4 hours, while macromolecules constituting the hydrogel are not degraded, and if the hydrogel is concentrated, the degradation rate of the hydrogel is slower. As shown in FIG. 10, the two degradants were transcribed after 4 hours of degradation, and the degradation was compared by measuring the fluorescence intensity. Negative control is that no degradation product is added under the same reaction condition, and positive control is that macromolecules obtained by adding hairbrush primer PCR without degradation treatment under the same reaction condition. The result shows that the functional gene wrapped by the hydrogel is not easy to degrade compared with the free linear DNA, so that the hairbrush primer PCR product can last for a longer time when being expressed in a cell-free system, and the expression quantity is theoretically higher than that of the isoconcentration linear DNA.

EXAMPLE 3 integration of different modified nucleotides and efficiency of amplification Studies

1) Primer extension with modified nucleoside triphosphates:

primer FAM-T1-R (1. mu. mol/L, Table 1) with the 5' end being fluorescence modified by FAM was annealed to DNA template T1 (2. mu. mol/L, Table 1) in 2 XOneTaq polymerase buffer (reaction conditions: 95 ℃ C., 5min, gradually cooled to room temperature and then placed on ice for 5 min). FAM was modified at the 5-terminus of the primer, and the band observed was the extension product.

The annealed template/primer mix (0.5. mu. mol/L) was incubated with native or modified deoxyribonucleoside triphosphate dNTPs or ribonucleoside triphosphate NTPs (0.5. mu. mol/L each) in 1 XOneTaq polymerase buffer containing 1. mu. mol/L SFM4-3 polymerase for 12h at 50 ℃.

When testing the ability of the remaining nucleotides, except those containing the modified nucleotides, to be extended with dNTPs, the experimental set was set as follows: (ii) 0.5. mu. mol/L of 5-pro-paramylamino-dCTP, no dCTP, ((iii) 0.5. mu. mol/L of 2 '-fluoro-CTP, ((iv) no 2' -fluoro-CTP), ((iv) 0.5. mu. mol/L of 5-pro-paramylamino-dCTP + 0.5. mu. mol/L of 2 '-fluoro-UTP), ((iii) no 5-pro-paramino-dCTP and 2' -fluoro-UTP, and (iv) natural dNTPs as the rest (FIG. 11A).

When testing the ability of the remaining nucleotides, except those containing the modification, to be extended with NTPs, the experimental groups were set as follows: adding 0.5 mu mol/L of 5-paramylono-dCTP, no 5-paramylono-dCTP, adding 0.5 mu mol/L of 2 '-fluoro-CTP, no 2' -fluoro-CTP, adding 0.5 mu mol/L of 5-paramylono-dCTP +0.5 mu mol/L of 2 '-fluoro-UTP, and no 5-paramylono-dCTP and 2' -fluoro-UTP, and the rest is natural NTPs (figure 11B).

Gel electrophoresis was performed using 18% denatured urea gel (containing 8mol/L urea). The extension band containing the 5' -FAM primer can be directly presented by a gel camera. The results are shown in FIG. 11. The results showed that the full-length nucleic acids could be obtained from all groups (i), (iii) and (iv), indicating that the SFM4-3 polymerase could recognize both base-modified and sugar-modified nucleotides, while the full-length nucleic acids could not be obtained from all groups (i) and (iv), indicating that both base-modified and sugar-modified nucleotides could be incorporated into the nucleic acids.

2) PCR of band-modified nucleic acids with different polymerases:

(ii) use of SFM4-3 polymerase

Template T1(4nmol/L, 75bp, Table 1), forward primer T1-F (0.5. mu. mol/L, Table 1), reverse primer T1-R (0.5. mu. mol/L, Table 1), native dGTP, dTTP, dATP and modified 5-proparylamino-dCTP each 0.4mmol/L, 0.1% BSA and 400nmol/L SFM4-3 polymerase were added to 1 XSF polymerase buffer to perform PCR (reaction conditions: 94 ℃ 2 min; 94 ℃ 30s, 50 ℃ 1min, 55 ℃ 2min, 15 cycles; 55 ℃ 5 min). Gel electrophoresis was performed on 6% PAGE gels. As shown in fig. 12A.

② use of Q5 polymerase

Template T1(4nmol/L, 75bp, Table 1), forward primer T1-F (0.3. mu. mol/L, Table 1), reverse primer T1-R (0.3. mu. mol/L, Table 1), and Q5 polymerase (20U/mL, each 0.2mmol/L of native dGTP, dTTP, dATP, and 5-proparylamino-dCTP containing a modification) were added to 1 XQ 5 polymerase buffer to perform PCR (reaction conditions: 98 ℃ 2 min; 98 ℃ 10s, 70 ℃ 30s, 72 ℃ 1min, 20 cycles; 72 ℃ 5 min). Gel electrophoresis was performed on 6% PAGE gels. As shown in FIG. 12B, NC is a PCR system without enzyme under the same conditions.

③ use OneTaq polymerase

Template T1(4nmol/L, 75bp, Table 1), forward primer T1-F (0.2. mu. mol/L, Table 1), reverse primer T1-R (0.2. mu. mol/L, Table 1), native dGTP, dTTP, dATP and modified 5-proparylamino-dCTP each 0.2mmol/L, 50U/mL OneTaq polymerase were added to 1 XOneTaq polymerase buffer solution to perform PCR (reaction conditions: 94 ℃ 2 min; 94 ℃ 30s, 66 ℃ 1min, 68 ℃ 1min, 30 cycles; 68 ℃ 5 min). Gel electrophoresis was performed using 6% PAGE gel. As shown in fig. 12B.

Fourthly, using Taq polymerase

Template T1(2nmol/L, 75bp, Table 1), upstream primer T1-F (0.2. mu. mol/L, Table 1), downstream primer T1-R (0.2. mu. mol/L, Table 1), native dGTP, dTTP, dATP and modified 5-propergylamino-dCTP each 0.2mmol/L, 25U/mL Taq polymerase were added to 1 XThermopol reaction buffer to perform PCR (reaction conditions: 95 ℃ 2 min; 95 ℃ 30s, 54 ℃ 1min, 68 ℃ 1min, 30 cycles; 68 ℃ 5 min). Gel electrophoresis was performed on 6% PAGE gels. As shown in fig. 12B.

3) Nucleotides with both base modification (5-propargylamino-dCTP) and glycosyl modification (2' -fluoro-UTP) were simultaneously incorporated by SFM4-3 polymerase PCR:

the procedure was as above for SFM4-3 polymerase PCR conditions, replacing dTTP with 2' -fluoro-UTP. As shown in FIG. 13, the results showed that SFM4-3 can incorporate both base-modified and glycosyl-modified nucleotides by PCR.

4) The base modified nucleotide (5-proparylamino-dCTP) and the unnatural base pair (dTPT3/dNaM) are simultaneously incorporated by SFM4-3 polymerase PCR:

to 1 XOneTaq polymerase buffer was added template TC6(16nmol/L, dNaM ═ X, Table 1), forward primer TC6-F (2.5. mu. mol/L, Table 1), reverse primer TC6-R (2.5. mu. mol/L, Table 1), native dGTP, dTTP, dATP and modified 5-proparamino-dCTP each 0.3mmol/L, 1.2mmol/L MgSO4, dPT 3-biotin (15. mu. mol/L), dNaM (0.1mmol/L), 2.42U/mL Deep Vent polymerase, 140nmol/L SFM4-3 polymerase, and PCR was carried out (reaction conditions: 94 ℃ 2 min; 94 ℃ 30s, 50 ℃ 1min, 55 ℃ 4 min; 15 cycles; 55 ℃ 10 min).

The incorporated dTPT3 carries a biotin (biotin) label, which binds to streptavidin and allows the PCR product to be tested for the presence of unnatural base pairs (dTPT3/dNaM), while the modified amino group can react with biotin-N-succinimidyl ester (NHS-biotin) and subsequently bind to streptavidin. And (3) respectively reacting the PCR product with streptavidin and NHS-biotin:

purifying a PCR product by using a DNA purification kit, adding streptavidin (0.1mg/mL) and incubating at 37 ℃ for 2 h;

purifying the PCR product by using a DNA purification kit, adding 5.6mmol/L NHS-biotin into 1 XPBS (pH 8.5) buffer solution, incubating at 37 ℃ for 12h, and purifying by using the DNA purification kit again;

③ incubating the product obtained by the reaction and purification of the previous step with NHS-biotin with streptavidin (0.1mg/mL) at 37 ℃ for 2 h;

and fourthly, performing gel electrophoresis by using 6 percent PAGE gel. The results are shown in FIG. 14A (p: PCR product, S1: dTPT 3-biotin-streptavidin conjugate on PCR product, S2: modified amino and NHS-biotin reactant on PCR product, t: S1 and S2 and streptavidin conjugate). The results show that SFM4-3 can simultaneously integrate a modified nucleotide at the base and an unnatural base pair by PCR.

5) The simultaneous incorporation of sugar-based modified nucleotides (2' -fluoro-CTP) and unnatural base pairs (dTPT3/dNaM) was performed by SFM4-3 polymerase PCR:

to 1 XTaq polymerase buffer was added template TC6(16nmol/L, dNaM ═ X, Table 1), forward primer TC6-F (2.5. mu. mol/L, Table 1), reverse primer TC6-R (2.5. mu. mol/L, Table 1), native dGTP, dTTP, dATP and modified 2' -fluoro-CTP 0.5mmol/L, 2mmol/L MgSO4, 0.1mmol/L MnCl ₂ dTPT3-biotin (0.1mmol/L), dNaM (0.1mmol/L), 2.42U/mL Deep Vent polymerase, 280nmol/L SFM4-3 polymerase, and PCR (reaction conditions: 94 ℃ for 2 min; 94 ℃ for 30s, 50 ℃ for 1min, 55 ℃ for 1h, cycle for 15 times; 55 ℃ for 1 h).

The incorporated dTTP 3 carries a biotin label, biotin binds to streptavidin and the PCR product can be tested for the presence of unnatural base pairs (dTPT 3/dNaM). The PCR product was purified with a DNA purification kit, incubated at 37 ℃ for 2 hours with streptavidin (0.1mg/mL), and subjected to gel electrophoresis with 6% PAGE gel. The results are shown in FIG. 14B (p: PCR product, s: dTPT3-biotin on PCR product with streptavidin conjugate). The results show that SFM4-3 can simultaneously integrate nucleotides with modifications and unnatural base pairs on the glycosyl group by PCR.

6) Simultaneous incorporation of ribose and deoxyribose nucleotides by SFM4-3 polymerase PCR:

template T1(20nmol/L, Table 1), forward primer T1-F (2. mu. mol/L, Table 1), reverse primer T1-R (2. mu. mol/L, Table 1), native dTTP, GTP, ATP and modified 5-proparylamino-dCTP each 1mmol/L, 0.1% TritonX-100, 0.1% BSA, 2mmol/L MgCl, were added to 1 XSF polymerase buffer ₂ 400nmol/L SFM4-3 polymerase or SF-WT wild-type polymerase at equal concentration, and performing PCR (reaction conditions: 94 deg.C for 2min, 94 deg.C for 30s, 49 deg.C for 1 min)1h at 50 ℃, and circulating for 15 times; 50 ℃ for 2 h). Gel electrophoresis was performed on 6% PAGE gels. The results are shown in FIG. 15. The results showed that SFM4-3 was able to synthesize a nucleic acid strand hybridized between deoxyand non-deoxynucleotides by PCR, whereas the wild-type polymerase SF-WT was not. SFM4-3 can provide conditions for synthesizing hybrid hydrogel scaffolds.

Example 4 PCR with Q5 polymerase to precisely control amino modification open density and Linker selection

The dCTP or dGTP of the sequences of the templates T3 and T4(200bp, Table 1) are separated by 5 and 10 bases except for the primer. PCR was performed using the templates T3 and T4 to amplify DNA containing 5-proparylamino-dCTP at different densities in a PCR system: 1 XQ 5 polymerase buffer, 1 XQ 5 High GC Enhancer, 0.2mmol/L each of native dGTP, dTTP, dATP and 5-proparylamino-dCTP containing a modification, upstream primer T3-F (0.5. mu. mol/L, Table 1), downstream primer T3-R (0.5. mu. mol/L, Table 1), T3, T4 (5.5 nmol/L each, Table 1), 20U/mL Q5 polymerase. The system is used for PCR reaction (reaction conditions: 98 ℃ for 2min, 98 ℃ for 10s, 70 ℃ for 30s, and 72 ℃ for 1min, cycle for 20 times, and 72 ℃ for 5 min). Gel electrophoresis was performed on 6% PAGE gels.

In 1 XPBS (pH 8.5) environment, adding different density 5-propragylamino-dCTP DNA products each 1 u mol/L, and NHS-FAM (5-carboxyl fluorescein succinimide ester) (200 u mol/L) at 37 degrees C reaction for 12 h. Gel electrophoresis was performed on 6% PAGE gels. The results are shown in fig. 16, which shows that the stripe luminance is: the intensity of the DNA product band of 5-prolylamino-dCTP separated by 5 bases is stronger than that of 5-prolylamino-dCTP separated by 10 bases, which indicates that the PCR is used for accurately controlling the density of amino modification by Q5 polymerase.

Referring to the method of example 1, two kinds of Linker NHS-DBCO and NHS-TCO were used to react with the DNA having amino group modifications at the base prepared in example 1, and it is seen from FIG. 17A that the bands were finer after reaction with NHS-DBCO than after reaction with NHS-TCO, indicating more complete reaction, so that it is preferable to use NHS-DBCO as the Linker. FIG. 17B shows the reaction of DNA with DBCO attached to a primer with an azide modification at the 5-terminus.

Example 5 detection of fidelity of 5-propragylamino-dCTP by PCR Using Q5 polymerase

1. PCR was performed on 5-proparylamino-DNA. PCR system (final concentration): dATP, dGTP, dTTP, 5-propagylamino-dCTP each 0.2mmol/L, Q5 polymerase 20U/ml, 1 XQ 5 polymerase buffer, Biotin-T1(4nmol/L), upstream primer T1-F (0.3. mu. mol/L, Table 1), downstream primer T1-R (0.3. mu. mol/L, Table 1). The system was used to perform PCR (reaction conditions: 98 ℃ for 2min, 98 ℃ for 10s, 70 ℃ for 30s, and 72 ℃ for 1min, cycle 20 times, 72 ℃ for 5 min). The PCR product (5-procargylamino-DNA) was purified and detected by 6% polyacrylamide gel electrophoresis and stained with Cyber gold.

2. Fidelity detection of PCR on 5-proparylamino-DNA

Removing a Biotin-T1 template: and (3) taking 15 mu L of streptavidin magnetic beads, washing the streptavidin magnetic beads for three times by using 5 × binding and washing buffer, washing the streptavidin magnetic beads for one time by using 1 × binding and washing buffer, adding the purified PCR product, shaking the PCR product for 2 hours at 37 ℃, standing the PCR product on a magnetic frame for 30 minutes, and then sucking the supernatant for purification.

Secondly, the purified 5-propagylamino-DNA product is subjected to PCR amplification by using dNTPs, and the template of a PCR system is the purified 5-propagylamino-DNA product, 0.2mmol/L of dNTPs, 20U/mL of Q5 polymerase, 1 XQ 5 polymerase buffer solution, an upstream primer T1-CL-F (0.5 mu mol/L, table 1) and a downstream primer T1-CL-R (0.5 mu mol/L, table 1).

Cloning and sequencing: the PCR product was digested with EcoRI and HindIII and inserted into the similarly digested pUC19 plasmid. The enzymatic ligation products were chemically transformed into E.coli XL1-Blue cells, seeded on ampicillin-containing agar plates and incubated overnight at 37 ℃. And selecting a single colony for PCR, sending the positive clone to sequencing, comparing the sequencing result with the original T1 template, and observing whether a mutation condition exists. The results are shown in FIG. 18, where 9 samples were tested and the middle 29 bases (boxes) had no mutations.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Claims

1. A method for preparing a non-natural nucleic acid hydrogel, which is characterized by comprising the following steps: the method comprises the following steps:

(1) preparation of upstream and downstream 'hairbrush' primers:

a. the method comprises the following steps of (1) doping mateable 5-position propargylamine-containing modified artificial nucleotide of a base part into a polymerase chain reaction system to prepare a nucleic acid fragment containing an amino linking arm on a part of bases;

c. designing and synthesizing an upstream primer and a downstream primer according to the sequence of the target fragment, and modifying groups capable of generating click chemistry reaction with the joint at the 5' ends of the upstream primer and the downstream primer;

(2) preparation of non-natural nucleic acid hydrogel:

2. The method for producing the non-natural nucleic acid hydrogel according to claim 1, wherein:

the polymerase in the polymerase chain reaction system in the step a is any one of Taq DNA polymerase, OneTaq DNA polymerase, Q5 DNA polymerase or SFM 4-3.

3. The method for producing the non-natural nucleic acid hydrogel according to claim 1, wherein:

when the polymerase is SFM4-3, the artificial nucleotide which can be paired and contains propargylamine-modified at the position 5 of the base part is doped into a polymerase chain reaction system, the artificial nucleotide which can be paired and contains fluorine-containing group modified at the position 2 of the glycosyl part is doped into the polymerase chain reaction system, the nucleic acid fragment containing amino group modification on the base and fluorine-containing group modification on the glycosyl is prepared, and then the subsequent reaction is carried out;

and/or, doping an unnatural base dTPT3 and dNaM in a polymerase chain reaction system to prepare a nucleic acid fragment containing an unnatural base pair, and then carrying out subsequent reaction;

the nucleotides in the polymerase chain reaction system are deoxyribonucleotide mixtures, or ribonucleotide mixtures, or deoxyribonucleotide/ribonucleotide mixtures.

4. The method for producing a non-natural nucleic acid hydrogel according to claim 1, wherein:

the click chemistry joint in the step b is selected from a NHS-DBCO joint or a NHS-TCO joint;

and c, the group capable of carrying out click chemistry reaction with the joint is an azide group.

5. The method for producing the non-natural nucleic acid hydrogel according to claim 1, wherein:

the incorporation in step a is by complete or partial substitution of one or more of the natural nucleotides;

the length of the nucleic acid fragment in the step a is 50-100 bp.

6. The method for producing the non-natural nucleic acid hydrogel according to claim 1, wherein:

the molar ratio of the nucleic acid fragment to the click chemical linker in the step b is 1: 1000-1500;

the reaction conditions in the step b are that the temperature is 35-40 ℃ and the time is 10-15 h;

the reaction in the step b is carried out in a PBS buffer system with the pH value of 8.4-8.6.

7. The method for producing the non-natural nucleic acid hydrogel according to claim 1, wherein:

the molar ratio of the nucleic acid fragment to the upstream primer and the downstream primer in the step d is 8-12: 1;

the reaction system in the step d also contains NaCl and Tween 20; the composition is as follows: 0.8-1.2 mol/L NaCl, 0.04-0.06% Tween20, 2-4 mu mol/L nucleic acid fragment, and 20-40 mu mol/L5-end upstream primer or downstream primer;

the reaction conditions in the step d are that the temperature is 45-55 ℃ and the time is 10-15 h;

the reaction in step d is carried out in a PBS buffer system with the pH value of 7.3-7.5.

8. The method for producing a non-natural nucleic acid hydrogel according to claim 1, wherein:

the concentration conditions in step (2) are as follows: centrifuging at the rotating speed of 5000-7000 rpm for 25-35 min;

the high-temperature annealing operation in the step (2) is as follows: heating at 95 deg.C for 5min, and gradually cooling to room temperature.

9. A non-natural nucleic acid hydrogel, comprising: prepared by the method of any one of claims 1 to 8.

10. Use of the non-natural nucleic acid hydrogel of claim 9 in the biomedical field.