CN114790471B

CN114790471B - Non-natural nucleic acid hydrogel and preparation method and application thereof

Info

Publication number: CN114790471B
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: 2023-08-18
Anticipated expiration: 2041-01-26
Also published as: CN114790471A

Abstract

The invention discloses a non-natural nucleic acid hydrogel and a preparation method and application thereof, and belongs to the technical field of biological materials. The method of the invention operates as follows: the method comprises the steps of adding a pairing artificial nucleotide containing propargylamine group at the 5 th position of a base part into a polymerase chain reaction system to prepare a nucleic acid fragment containing an amino linking arm on part of the base, carrying out click chemical reaction with a primer containing a corresponding group at the 5' end respectively after carrying out click chemical reaction with a click chemical linker to obtain a brush primer, carrying out PCR amplification by using the nucleic acid fragment or plasmid containing the target fragment as a template through the brush primer, and carrying out high-temperature annealing after amplification product concentration. The method provided by the invention is simple and easy, has high universality, and the obtained hydrogel can protect functional DNA (deoxyribonucleic acid) from being degraded in serum, so that the defect of the traditional DNA hydrogel is overcome, and the hydrogel has the advantages of higher stability, controllable crosslinking degree, carrying genetic information and the like, and has a 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

Hydrogels are a common material, wherein part of the hydrogels have good biocompatibility, adjustable biodegradability and controllable mechanical properties, and have important application values in the fields of chemical and biomedical engineering. With the deep research of the hydrogel field, a more accurate synthesis method, more sensitive stimulus response and richer functionalities have become main research directions and targets in the development process of novel application type hydrogels.

DNA is the core genetic material of the life system, leading to biological development and vital functions. From the material chemistry perspective, DNA is a natural biological polymer and has the characteristic that synthetic polymers cannot be compared. For example: the base complementary pairing characteristic ensures that the DNA has accurate and efficient self-assembly capability; the DNA sequence is various and adjustable, the structure is accurate and controllable, and the stimulation response is rich; natural evolution endows organisms with rich and diverse biological enzymes, and can accurately operate DNA at a molecular level; the DNA has good biocompatibility and biodegradability.

In recent years, scientists have synthesized a variety of DNA functional polymeric materials using DNA as a building block. Among them, DNA hydrogel is an important DNA material, and is a three-dimensional polymer network constructed by using DNA as a structural element, and has been vigorously developed in recent years. The DNA hydrogel not only utilizes the skeleton structure of the hydrogel, but also retains the biological function of DNA, realizes 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. For example, preformed DNA building blocks (X, Y, T type DNA) are interacted with complementary DNA strands by controlling the enzyme or pH. DNA hydrogels can also be prepared by PCR amplification using Y-shaped DNA primers or multiple primer rolling circle replication. However, these hydrogels are formed by hydrogen bonding between bases, have low stability, require special designs for sequences, and have limited controllability of many properties (such as degree of 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, a primary object of the present invention is to provide a method for preparing a non-natural nucleic acid hydrogel, which is a method for efficiently preparing a nucleic acid hydrogel with more excellent properties by using a non-natural nucleic acid scaffold.

It is another object of the present invention to provide a non-natural nucleic acid hydrogel obtained by the above preparation method.

It is a further object of the present invention to provide the use of the above-described non-natural nucleic acid hydrogel.

The aim of the invention is achieved by the following technical scheme:

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

(1) Preparation of upstream and downstream "brush" primers:

a. incorporating a pairing-able artificial nucleotide containing propargylamine group at the 5 th position of a base part into a polymerase chain reaction system to prepare a nucleic acid fragment containing an amino connecting arm on part of the base;

b. c, reacting the nucleic acid fragment obtained in the step a with a click chemistry 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 the 5' ends of the upstream primer and the downstream primer to form a group which can perform click chemical reaction with a linker;

d. c, carrying out click chemistry reaction on the nucleic acid fragments containing the linker on part of the nucleotides obtained in the step b and the upper and lower primers containing the modification group at the 5' end obtained in the step c respectively to obtain upper and lower hairbrush primers;

(2) Preparation of non-natural nucleic acid hydrogels:

and (3) taking a nucleic acid fragment or plasmid containing the target fragment as a template, carrying out PCR amplification by using the brush primers on the upstream and downstream obtained in the step (1), concentrating an amplification product, and annealing at a high temperature 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 paired artificial nucleotide modified by fluorine-containing groups on the No. 2 locus of the glycosyl part can be doped into a polymerase chain reaction system to prepare a nucleic acid fragment modified by the fluorine-containing groups on the glycosyl and the amino groups on the base, and then subsequent reaction is carried out.

When the polymerase is SFM4-3, unnatural base dTTT 3 and dNaM can be doped into a polymerase chain reaction system to prepare a nucleic acid fragment containing unnatural base pairs, and then subsequent reaction is carried out.

The nucleotides in the polymerase chain reaction system can be deoxyribonucleotide mixtures, or ribonucleotide mixtures, or deoxyribonucleotide/ribonucleotide mixtures, so as to prepare corresponding nucleic acid fragments, and then carrying out subsequent reactions.

The incorporation in step a may be carried out by completely or partially substituting one or more of the natural nucleotides, preferably with a nucleic acid content of 10% to 40% of the amino-linked arm-containing nucleic acids on the bases of the resulting nucleic acid fragment.

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

The click chemistry linker described in step b may be selected from NHS-DBCO (diphenylcyclooctyne-succinimidyl ester) linker, NHS-TCO (trans cyclooctene-succinimidyl ester) linker; NHS-DBCO linkers are preferred.

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

The reaction conditions in step b are preferably 35 to 40℃for 10 to 15 hours, more preferably 37℃for 12 hours.

The reaction described in step b is preferably carried out in a PBS buffer system having a ph=8.4 to 8.6.

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

The reaction system in the step d also contains NaCl and Tween20; more preferred compositions are as follows: 0.8-1.2 mol/L NaCl, 0.04-0.06% Tween20, 2-4 mu mol/L nucleic acid fragment, 20-40 mu mol/L5 end upstream primer or downstream primer; the most preferred 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 described in step c which is capable of undergoing click chemistry with the linker is preferably an azide group.

The reaction conditions in step d are preferably 45 to 55℃for 10 to 15 hours, more preferably 50℃for 12 hours.

The reaction described in step d is preferably carried out in a PBS buffer system having a ph=7.3 to 7.5.

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

The high temperature annealing operation described in step (2) is preferably as follows: after heating at 95℃for 5min, it was gradually cooled to room temperature.

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

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 modification on base by enzyme method, provides covalent binding site, and solves the problem of low stability of DNA hydrogel by introducing covalent bond. The nucleotide with modified base is cheap and easy to obtain, can be identified by various nucleic acid polymerases, and provides a convenient preparation method for hydrogel nucleic acid skeleton formation. Meanwhile, by controlling the sequence of the nucleic acid skeleton, the properties of the formed hydrogel such as the degree of density, mechanical property, stability and the like can be conveniently controlled and optimized. Based on the above, the invention also uses a mutant strain SFM4-3 of the directed evolution polymerase-Taq DNA polymerase Stofel fragment, integrates nucleotide modified on a base and a glycosyl and introduces unnatural base pair (dTTT 3/dNAM) simultaneously by PCR, and provides more connecting sites for introducing covalent bonds, thereby increasing the potential application of the hydrogel (for example, covalent bonds are introduced by modification on the base for forming the hydrogel, and covalent bonds are introduced by modification on the glycosyl for connecting sustained release drug molecules). Meanwhile, the polymerase SFM4-3 can synthesize a hybrid nucleic acid chain of deoxyribonucleoside and ribonucleotide, and provides a new method for forming hybrid hydrogel of deoxynucleotide and non-deoxynucleotide.

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

the invention explores 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 not only overcomes the defects of the traditional DNA hydrogel, but also has the advantages of higher stability, controllable crosslinking degree, carrying genetic information and the like, and the potential application of the DNA hydrogel is increased on the basis. The nucleic acid hydrogel prepared by using the nucleic acid modified by the base 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. the controllability of properties such as degree of density, mechanical properties, etc. is limited.

Increasing the potential applications of DNA hydrogels:

1. a plurality of covalent bond introducing sites are provided, 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 the nucleic acid hydrogel;

3. can be used for wrapping functional DNA, so that the functional DNA is not easy to degrade in serum;

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

5. The introduction of covalent bond sites can be precisely controlled, so that the degree of cross-linking of the hydrogel can be controlled.

Drawings

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

FIG. 2 is a graph showing the result of electrophoresis of PCR amplification products of different brush primers in example 1; wherein, diagram a: lane M is Marker, lane 1 is the normal primer PCR result, lane 2 is the Broccoli "brush primer" PCR result, fig. B: lane M is Marker, lane 1 is GFP "brush primer" PCR results.

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

FIG. 4 is a graph showing the fluorescence microscopy results after annealing and concentrating the non-natural nucleic acid hydrogel containing the Broccoli gene prepared in example 1.

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

FIG. 6 is a graph showing the real-time detection of transcription of brush PCR products containing the Broccoli gene by the microplate reader of example 2.

FIG. 7 is a diagram showing the expression of green fluorescent protein in a cell-free system of the brush PCR product containing GFP gene in example 2; wherein, 1 is a template-free negative control group; 2 is a brush PCR negative control product group; 3 is a linear template positive control group; 4 is an azide modified template positive control group; 5 is brush PCR product experimental group I (15 ng/. Mu.l); 6 is brush PCR product experimental group II (45 ng/. Mu.l); 7 is brush PCR product experimental group III (75 ng/. Mu.l).

FIG. 8 is a graph showing the comparison of fluorescent intensity of a brush PCR product containing GFP gene with that of linear DNA of equal concentration after expressing proteins in a cell-free system in example 2.

FIG. 9 is a diagram showing degradation of brush PCR products containing the Broccoli gene in fetal bovine serum in example 2.

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

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

FIG. 12 is a diagram of electrophoresis of nucleotide modified by PCR introduction of base (5-procyanino-dCTP) with different polymerases; wherein, the diagram A is SFM4-3 polymerase PCR product, and the diagram B is Q5, oneTaq, taq polymerase PCR product.

FIG. 13 shows a nucleotide electrophoresis pattern of SFM4-3 polymerase PCR incorporating both base modification (5-propergylamino-dCTP) and glycosyl modification (2' -fluoro-UTP).

FIG. 14 is an electrophoretogram of SFM4-3 polymerase PCR incorporating both base modified (5-propertyiamino-dCTP) nucleotides and unnatural base pairs (dTTT 3/dNaM); wherein, FIG. A is a PCR product (p: PCR product, S1: dTTT 3-biotin and streptavidin conjugate on the PCR product, S2: modified amino and NHS-biotin reactants on the PCR product, t: S1 and S2 and streptavidin conjugate), FIG. B is an electrophoresis pattern (p: PCR product, S: dTTT 3-biotin and streptavidin conjugate on the PCR product) of SFM4-3 polymerase PCR with simultaneous incorporation of glycosyl modified (2' -fluoro-CTP) nucleotides and unnatural base pairs (dTTT 3/dNAM).

FIG. 15 is a diagram showing electrophoresis of simultaneous incorporation of ribose and deoxyribose nucleotides by SFM4-3 polymerase PCR.

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

FIG. 17 is a graph showing the results of the reaction of the DNA having amino group modification with the base prepared in example 1 using different linkers in example 4; wherein, the graph A is a graph of the reaction results of different Linker and DNA containing modified base, 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 sequencing results 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 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 relates.

Materials or reagents involved in the following examples:

SFM4-3 polymerase, SF-WT polymerase: the literature "Chen T, hongdilokkul 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-procyanino-dCTP was purchased from: the model of the UK Hua Ren technology Co.Ltd is HR-00104022

2' -fluoro-CTP was purchased from: the model of the UK Hua Ren technology Co.Ltd is HR-00104018

2' -fluoro-UTP was purchased from: the model of the UK Hua Ren technology Co.Ltd is HR-00104020

2' -methoxy-GTP was purchased from: the model of the UK Hua Ren technology Co.Ltd is HR-00104003

dNTP sets were purchased from: new England Biolabs model N0446S

rNTP kit was purchased from: new England Biolabs model N0450S

dTTT 3-biotin, dNAM, already known in the literature "Li L, degardin M, lavergne T, et al Natural-like

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) 826-829. "A method for producing a polypeptide of formula I

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 Yitao Biotechnology Co., ltd, model bcd6

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

DHFBI was purchased from: glpbio Co., U.S. model GC30098

NHS-FAM was purchased from: shanghai Tuo Biotechnology Co., ltd., model HY-15938

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

Cyber Gold was purchased from: hundred Fluor Biotech Co., ltd., model TJ702

EcoRI was purchased from: the model number of the Siemens technology company is FD0274

HindIII was purchased from: the model number of the Siemens technology company is FD0504

T4 ligase was purchased from: new England Biolabs model M0202S

1X binding and washing buffer formulation: 10mmol/L Tris-HCl (pH 7.4), 1mol/LNaCl,0.1% Tween20,1mmol/L EDTA

Magnesium acetate was purchased from: hadamard products Co., ltd., model 01023011

Calcium folinate hydrate salts were purchased from: hadamard products Co., ltd., model 25231A

Sodium pyruvate was purchased from: hadamard products Co., ltd., model 01009805

Ammonium hydroxide solution was purchased from: shanghai Miclin Biochemical technology Co., ltd., model A801005

Oxalic acid was purchased from: hadamard products Co., ltd., model 01023951

HEPES was purchased from: hadamard products Co., ltd., model 01110191

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

1, 4-diaminobutane was purchased from: hadamard products Co., ltd., model 01007891

Spermidine was purchased from: hadamard products Co., ltd., model S02660

The monopotassium phosphate (enol) pyruvate (PEP) is purchased from: sigma-Aldrich model P7127

Potassium hydroxide was purchased from: shanghai microphone Lin Biochemical technology Co., ltd., model P822103

L-alanine was purchased from: hadamard products Co., ltd., model 01088967

L-arginine was purchased from: hadamard products Co., ltd., model 01111537

L-asparagine was purchased from: hadamard products Co., ltd., model 01107585

L-aspartic acid was purchased from: hadamard products Co., ltd., model 01089459

L-cysteine was purchased from: hadamard products Co., ltd., model 01083581

L-glutamic acid was purchased from: shanghai Shaoshao far reagent Co., ltd., model SY008812

L-Glutamine was purchased from: hadamard products Co., ltd., model 01089485

Glycine was purchased from: hadamard products Co., ltd., model 01088948

L-histidine was purchased from: hadamard products Co., ltd., model 01108001

L-isoleucine was purchased from: hadamard products Co., ltd., model TZL36951

L-leucine was purchased from: hadamard products Co., ltd., model 01096937

L- (+) -lysine was purchased from: hadamard products Co., ltd., model 01089505

L-methionine was purchased from: hadamard products Co., ltd., model 01100470

L-phenylalanine was purchased from: hadamard products Co., ltd., model 01100850

L proline was purchased from: hadamard products Co., ltd., model 01025290

L-serine was purchased from: hadamard products Co., ltd., model 01089010

L-threonine was purchased from: hadamard products Co., ltd., model 01109021

L tryptophan was purchased from: hadamard products Co., ltd., model 01109787

L-tyrosine was purchased from: hadamard products Co., ltd., model 01094035

L-Valine was purchased from: hadamard products Co., ltd., model 01109011

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 is purchased from: sigma-Aldrich model N6522

Adenine nucleoside triphosphates were purchased from: siemens Feishul technology Co, model R0481

Guanosine triphosphates were purchased from: siemens Feishul technology Co, model R0481

Cytosine nucleoside triphosphates were purchased from: siemens Feishul technology Co, model R0481

Uridine triphosphate was purchased from: siemens Feishul technology Co, model R0481

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

Coenzyme a hydrate was purchased from: sigma-aldrich model C4282

Dithiothreitol was purchased from: hadamard products Co., ltd., model 01064273

Dipotassium hydrogen phosphate was purchased from: shanghai Taitan technologies Co., ltd., model V900050

Monopotassium phosphate was purchased from: shanghai Taitan technologies Co., ltd., model G82821B

Yeast extracts were purchased from: shanghai Taitan technologies Co., ltd., model LP0021

Protein is purchased from: shanghai Taitan technologies Co., ltd., model LP0042

Sodium chloride was purchased from: shanghai Taitan technologies Co., ltd., model G81793H

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

TABLE 1 sequence names and sequences

EXAMPLE 1 preparation of unnatural nucleic acid hydrogels

(1) Obtaining a DNA comprising modified bases:

template T1 (4 nmol/L,75bp, table 1), upstream primer T1-F (0.3. Mu. Mol/L, table 1), downstream primer T1-R (0.3. Mu. Mol/L, table 1), natural dGTP, dTTP, dATP and modified 5-procyanino-dCTP each 0.2mmol/L,20U/ml Q5 polymerase were added to 1 XQ 5 polymerase buffer, and PCR reactions were performed (reaction conditions: 98℃2min;98℃10s, 70℃30s, 72℃1min, 20 cycles; 72℃5 min) to obtain PCR products containing modified 5-procyanino-dCTP at the bases, and purified using DNA purification kit.

(2) Preparing an upstream and downstream brush primer:

1. Mu. Mol/L of the base obtained in step (1) containing the modified PCR product of 5-procyanino-dCTP and 1mmol/L of NHS-DBCO were added to a 1 XPBS (pH 8.5) buffer, and after incubation at 37℃for 12 hours, 6% polyacrylamide gel electrophoresis was used to verify the reaction. The reaction product was centrifuged 6 times with 1 XPBS (pH 7.4) at 6000rpm using a 30kDa Amicon filter for 15min each.

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 (pH 7.4) buffer solution ₃ Broccoli-F (33. Mu. Mol/L, table 1) or N ₃ GFP-F (40. Mu. Mol/L, table 1), 5-terminalAzide modified downstream primer N ₃ Broccoli-R (33. Mu. Mol/L, table 1) or N ₃ GFP-R (40. Mu. Mol/L, table 1), after incubation at 50℃for 12h, gel electrophoresis with 6% PAGE gel was used to verify the reaction. The reaction product was centrifuged 6 times with 1 XPBS (pH 7.4) at 6000rpm using a 30kDa Amicon filter for 15min each. The manufacturing process is shown in fig. 1.

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

a template T2 (4 nmol/L, table 1), an upstream primer T2-F-P (0.5. Mu. Mol/L, table 1) containing a start code, a downstream primer T2-R-T (0.5. Mu. Mol/L, table 1) containing a stop code, 0.2. Mu. Mol/L dNTPs, and 25U/mL of Taq polymerase were added to a 1X thermo pol reaction buffer, and a PCR reaction was performed (reaction conditions: 95℃30s; 63℃30s; 68℃15 s; 30 cycles; 68℃5 min). The Broccoli gene carrying the start and stop codons was obtained by PCR. The PCR product was purified and used as a template for the next PCR.

The PCR reaction was performed by adding 37nmol/L, 0.2. Mu. Mol/L dNTP, the upstream primer T2-F-E containing EcoRI cleavage site (0.2. Mu. Mol/L, table 1), the downstream primer T2-R-H containing HindIII cleavage site (0.2. Mu. Mol/L, table 1) and 25U/mL Taq polymerase (reaction conditions: 95 ℃ C. 30s; 65 ℃ C. 1 min; 68 ℃ C. 20 s; 30 cycles; 68 ℃ C. 5 min) of the PCR product to 1X thermo pol reaction buffer. The Broccoli gene fragment and pUC57 plasmid with enzyme cutting site are cut, the Broccoli gene fragment and pUC57 plasmid are added into 1X FastDiget Green buffer solution, ecoRI and HindIII are added proportionally, after reacting for 12 hours at 37 ℃, the cut and purified fragment and vector are added into 1X T4 DNA ligase buffer solution according to the ratio of 3:1, the Broccoli gene and pUC57 plasmid are connected by T4 DNA ligase, after connecting for 12 hours at 16 ℃, the connecting product is transferred into DH5 alpha competent cells, and a large amount of plasmid containing the Broccoli gene is obtained.

(4) Hydrogel was prepared and concentrated:

preparation of hydrogels containing the Broccoli gene: 1nmol/L of template containing Broccoli gene in the previous step, 0.2mmol/L dNTP, 43nmol/L of each of "brush primer" upstream of Broccoli and "brush primer" downstream of Broccoli, 1mol/L NaCl,0.05% Tween20, 50U/mL OneTaq (94 ℃ C. 2min;94 ℃ C. 30s, 50 ℃ C. 1min, 68 ℃ C. 5min, 30 cycles; 68 ℃ C. 10 min) were added to 1 XOneTaq buffer, respectively. Gel electrophoresis was performed with 1% agarose to verify whether there was a large molecule trapped in the well, and the results are shown in FIG. 2A (1 is a negative control: ordinary primer PCR product, 2 is a brush primer PCR product).

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

The hydrogel containing the Broccoli gene was centrifuged at 6000rpm for 30min with a 30kDa Amicon filter and heated at 95℃for 5min before gradually cooling to room temperature to form a gel. As shown in fig. 3A, the macromolecules formed by PCR are combined and concentrated to form loose micelles; as shown in fig. 3B, the hydrogel was more dense after high temperature annealing, and after staining with Cyber Gold, significant fluorescence was seen under uv light, thus proving that the main component of the hydrogel was nucleic acid.

(6) Characterization of non-natural nucleic acid hydrogels:

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

After freeze-drying the hydrogel and surface plating, the fine structure inside was observed with a Scanning Electron Microscope (SEM), and it was found to be nanoflower-like, as shown in fig. 5.

EXAMPLE 2 use of unnatural nucleic acid hydrogels

1. Transcribable carried genetic information

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

2. Can express the carried genetic information

Expressing the brush PCR product containing GFP gene in a cell-free system of escherichia coli, and finally, the concentration of each reagent in the reaction system of the cell-free system: 1.2mmol/L adenine nucleoside triphosphate, 0.850mmol/L guanine nucleoside triphosphate, 0.850mmol/L urine purine nucleoside triphosphate, 0.850mmol/L cytosine nucleoside 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, 20 amino acids each 2mmol/L,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 at 30℃overnight. The experimental groups were set as follows: 1: a template-free negative control group; 2: brush PCR negative control product group (brush reaction system without PCR cross-linking); 3: linear template positive control (GFP-F, GFP-R (sequence with N) ₃ -GFP-F、N ₃ GFP-R) 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 was 40 ng/. Mu.l of PCR-purified product from primer amplification); 5: hairbrush PCR product experimental group I (hairbrush PCR product dialysate 15 ng/. Mu.l); 6: hairbrush PCR product experimental group II (hairbrush PCR product dialysate 45 ng/. Mu.l); 7: hairbrush PCR product experimental group III (hairbrush PCR product dialysate 75 ng/. Mu.l). As shown in FIG. 7, the difference between the expression of the brush hydrogel and the expression of the linear template, the azide-modified template and the brush negative control product is compared, so that the brush hydrogel can be used for in vitro transcription and translation and has better effect.

Two control experiments were set and three parallel controls were performed for each group, and as shown in fig. 8, cell-free protein expression systems containing and not containing brush PCR products were reacted at 37 ℃ for 20 minutes, and by monitoring fluorescence intensity, the brush hydrogel was again verified to be useful for transcription and translation in vitro and to have a good effect.

3. Can protect DNA carrying genetic information from degradation in fetal calf serum

Macromolecules obtained by PCR with the same nucleic acid concentration (274 ng/. Mu.l) and the Broccoli DNA amplified by the normal primer PCR are respectively placed in fetal bovine serum and degraded for 12 hours, and the two degradation products are analyzed by gel electrophoresis for strip fluorescence intensity every hour to obtain the change curves of the fluorescence intensity of the two strips. As shown in fig. 9, it can be seen that DNA was almost completely degraded in 4 hours under the same conditions, while the macromolecules constituting the hydrogel were not degraded yet, and if the hydrogel was concentrated, the degradation rate was slower. As shown in fig. 10, two degradation products were transcribed 4 hours after degradation, and the degradation was compared by detecting fluorescence intensity. The negative control is that no degradation product is added under the same reaction condition, and the positive control is that macromolecules which are not obtained by the PCR of the brush primer after degradation treatment are added under the same reaction condition. The results show that the functional genes wrapped by the hydrogel are less prone to degradation than free linear DNA, so that the brush primer PCR product can last longer when expressed in a cell-free system, and the expression quantity is theoretically higher than that of the linear DNA with equal concentration.

EXAMPLE 3 integration and amplification efficiency Studies of different modified nucleotides

1) Primer extension with modified nucleoside triphosphates:

the 5' -end FAM fluorescence-modified primer FAM-T1-R (1. Mu. Mol/L, table 1) was annealed to the DNA template T1 (2. Mu. Mol/L, table 1) in 2X OneTaq polymerase buffer (reaction conditions: 95 ℃,5min, gradually cooled to room temperature and then left on ice for 5 min). FAM was modified at the 5-terminus of the primer and the band seen was the extension product.

The annealed template/primer mixture (0.5. Mu. Mol/L) was incubated with natural or modified deoxyribonucleoside triphosphates 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 nucleotides other than the modified nucleotide to be extended with dNTPs, the experimental set was set up as follows: (1) incorporating 0.5. Mu. Mol/L5-procyanino-dCTP, (2) no dCTP, (3) incorporating 0.5. Mu. Mol/L2'-fluoro-CTP, (4) no 2' -fluoro-CTP, (5) incorporating 0.5. Mu. Mol/L5-procyanino-dCTP+0.5. Mu. Mol/L2'-fluoro-UTP, (6) no 5-procyanino-dCTP and 2' -fluoro-UTP, the remainder being natural dNTPs (FIG. 11A).

When testing the ability of nucleotides other than the modified nucleotide to extend with NTPs, the experimental set was set as follows: (1) incorporating 0.5. Mu. Mol/L5-procyanino-dCTP, (2) no 5-procyanino-dCTP, (3) incorporating 0.5. Mu. Mol/L2'-fluoro-CTP, (4) no 2' -fluoro-CTP, (5) incorporating 0.5. Mu. Mol/L5-procyanino-dCTP+0.5. Mu. Mol/L2'-fluoro-UTP, (6) no 5-procyanino-dCTP and 2' -fluoro-UTP, the remainder being natural NTPs (FIG. 11B).

Gel electrophoresis was performed with 18% modified urea gum (containing 8mol/L urea). The extension band containing the 5' -FAM primer can be presented directly by a gel reader. The results are shown in FIG. 11. The results show that the full length nucleic acid can be obtained in each of the experimental groups (1), (3) and (5), which means that the SFM4-3 polymerase can recognize the base modified and sugar modified nucleotides, whereas the truncated products can be obtained in each of the experimental groups (2) and (4), which do not obtain the full length nucleic acid, which means that the base modified and sugar modified nucleotides can be integrated into the nucleic acid.

2) PCR of different polymerases on modified nucleic acids:

(1) with SFM4-3 polymerase

A template T1 (4 nmol/L,75bp, table 1), an upstream primer T1-F (0.5. Mu. Mol/L, table 1), a downstream primer T1-R (0.5. Mu. Mol/L, table 1), each 0.4mmol/L of native dGTP, dTTP, dATP and modified 5-procyanino-dCTP, 0.1% BSA and 400nmol/L SFM4-3 polymerase were added to a 1 XSF polymerase buffer, and PCR reactions were performed (reaction conditions: 94℃2min;94℃30s, 50℃1min, 55℃2min, 15 cycles; 55℃5 min). Gel electrophoresis was performed with 6% page gels. As shown in fig. 12A.

(2) With Q5 polymerase

A template T1 (4 nmol/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), each 0.2mmol/L of native dGTP, dTTP, dATP and modified 5-procyanino-dCTP, 20U/mL Q5 polymerase were added to a 1 XQ 5 polymerase buffer, and PCR was performed (reaction conditions: 98℃2min;98℃10s, 70℃30s, 72℃1min, 20 cycles; 72℃5 min). Gel electrophoresis was performed with 6% page gels. As shown in FIG. 12B, NC is a PCR system without enzyme under the same conditions.

(3) By OneTaq polymerase

A PCR reaction (reaction conditions: 94℃2min;94℃30s, 66℃1min, 68℃1min, 30 cycles; 68℃5 min) was performed by adding template T1 (4 nmol/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), each 0.2mmol/L of native dGTP, dTTP, dATP and modified 5-procyanino-dCTP, 50U/mL OneTaq polymerase, to a 1 XOneTaq polymerase buffer. Gel electrophoresis was performed with 6% page gels. As shown in fig. 12B.

(4) By Taq polymerase

A template T1 (2 nmol/L,75bp, table 1), upstream primers T1-F (0.2. Mu. Mol/L, table 1), downstream primers T1-R (0.2. Mu. Mol/L, table 1), native dGTP, dTTP, dATP and modified 5-procyanino-dCTP each 0.2mmol/L,25U/mL Taq polymerase were added to a 1 XPol reaction buffer, and PCR reactions were performed (reaction conditions: 95℃2min;95℃30s, 54℃1min, 68℃1min, 30 cycles; 68℃5 min). Gel electrophoresis was performed with 6% page gels. As shown in fig. 12B.

3) Nucleotides incorporating both base modification (5-procyanino-dCTP) and glycosyl modification (2' -fluoro-UTP) by SFM4-3 polymerase PCR:

the method is similar to the SFM4-3 polymerase PCR conditions, and dTTP is replaced by 2' -fluoro-UTP. As shown in FIG. 13, the results showed that SFM4-3 could integrate both the nucleotides modified on the base and the nucleotides modified on the sugar group by PCR.

4) Simultaneously incorporating a base modified nucleotide (5-propargylamino-dCTP) and an unnatural base pair (dTTT 3/dNaM) by SFM4-3 polymerase PCR:

the template TC6 (16 nmol/L, dNaM=X, table 1), the upstream primer TC6-F (2.5. Mu. Mol/L, table 1), the downstream primer TC6-R (2.5. Mu. Mol/L, table 1), native dGTP, dTTP, dATP and modified 5-procyanino-dCTP were each 0.3mmol/L,1.2mmol/L MgSO4, dTTT 3-biotin (15. Mu. Mol/L), dNaM (0.1 mmol/L), 2.42U/mL Deep Vent polymerase, 140nmol/L SFM4-3 polymerase were added to a 1 XOneTaq polymerase buffer, and a PCR reaction was performed (reaction conditions: 94℃2min, 94℃30s, 50℃1min, 55℃4min, 15 cycles; 55℃10 min).

The incorporated dTTT 3 is labeled with biotin (biotin), which can bind to streptavidin, and the PCR product can be checked for the presence of unnatural base pairs (dTTT 3/dNAM), and the modified amino groups can be reacted with biotin-N-succinimidyl ester (NHS-biotin) to bind to streptavidin. The PCR products were reacted with streptavidin, NHS-biotin, respectively:

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

(2) the PCR product is purified by a DNA purification kit, 5.6mmol/L NHS-biotin is added into a 1 XPBS (pH 8.5) buffer solution, and the PCR product is further purified by the DNA purification kit after incubation for 12 hours at 37 ℃;

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

(4) gel electrophoresis was performed with 6% page gels. The results are shown in FIG. 14A (p: PCR product, S1: dTTT 3-biotin conjugate with streptavidin on PCR product, S2: modified amino group and NHS-biotin reactant on PCR product, t: S1 and S2 conjugate with streptavidin). The results show that SFM4-3 can simultaneously integrate base-modified nucleotides and unnatural base pairs by PCR.

5) Simultaneously incorporating a glycosyl modified nucleotide (2' -fluoro-CTP) and an unnatural base pair (dTTT 3/dNAM) by SFM4-3 polymerase PCR:

template TC6 (16 nmol/L, dNaM=X, table 1), upstream primer TC6-F (2.5. Mu. Mol/L, table 1), downstream primer TC6-R (2.5. Mu. Mol/L, table 1), native dGTP, dTTP, dATP and modified 2' -fluoro-CTP each 0.5mmol/L,2mmol/L MgSO4,0.1mmol/L MnCl were added to 1 XTaq polymerase buffer ₂ dTTT 3-biotin (0.1 mmol/L), dNAM (0.1 mmol/L), 2.42U/mL Deep Vent polymerase, 280nmol/L SFM4-3 polymerase, and performing PCR reaction (reaction conditions: 94 ℃ C. 2min;94 ℃ C. 30s, 50 ℃ C. 1min, 55 ℃ C. 1h, circulation)15 times; 55 ℃ for 1 h).

The doped dTTT 3 is provided with a biotin label, biotin can be combined with streptavidin, and whether the PCR product contains unnatural base pairs (dTTT 3/dNAM) can be detected. The PCR product was purified using a DNA purification kit, and after incubation at 37℃for 2 hours with streptavidin (0.1 mg/mL), gel electrophoresis was performed using a 6% PAGE gel. The results are shown in FIG. 14B (p: PCR product, s: dTTT 3-biotin conjugate on PCR product). The results show that SFM4-3 can simultaneously integrate glycosyl modified nucleotides and unnatural base pairs by PCR.

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

template T1 (20 nmol/L, table 1), upstream primer T1-F (2. Mu. Mol/L, table 1), downstream primer T1-R (2. Mu. Mol/L, table 1), native dTTP, GTP, ATP and modified 5-Proparylamino-dCTP each 1mmol/L,0.1% Triton X-100,0.1% BSA,2mmol/L MgCl were added to 1 XSF polymerase buffer ₂ A PCR reaction (reaction conditions: 94℃2min, 94℃30s, 49℃1min, 50℃1h, 15 cycles; 50℃2 h) was carried out with 400nmol/L SFM4-3 polymerase or an SF-WT wild-type polymerase of equal concentration. Gel electrophoresis was performed with 6% page gels. The results are shown in FIG. 15. The results show that SFM4-3 is capable of synthesizing nucleic acid strands heterozygous for deoxyand non-deoxynucleotides by PCR, whereas wild-type polymerase SF-WT is not. SFM4-3 can provide conditions for the synthesis of hybrid hydrogel backbones.

Example 4 precise control of Density of amino modifications and selection of Linker Using Q5 polymerase PCR

Templates T3, T4 (200 bp, table 1) apart from the primers, dCTP or dGTP of the sequence were separated by 5 and 10 bases, respectively. PCR was performed using templates T3 and T4 to amplify DNA containing 5-propargylamino-dCTP at different densities, with the PCR system: 1 XQ 5 polymerase buffer, 1 XQ 5 High GC Enhancer, native dGTP, dTTP, dATP and modified 5-procyanino-dCTP containing 0.2mmol/L each, 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. PCR was performed using the system (reaction conditions: 2min at 98 ℃, 10s at 98 ℃, 30s at 70 ℃, 1min at 72 ℃,20 cycles; 5min at 72 ℃). Gel electrophoresis was performed with 6% page gels.

In an environment of 1 XPBS (pH 8.5), 1. Mu. Mol/L each of the 5-propargylamino-dCTP DNA products of different densities was added and reacted with an excess of NHS-FAM (5-carboxyfluorescein succinimidyl ester) (200. Mu. Mol/L) at 37℃for 12h. Gel electrophoresis was performed with 6% page gels. The results are shown in fig. 16, and the results show that the band brightness is: the DNA product of 5-procyanino-dCTP was more bright than the DNA product of 5-procyanino-dCTP at intervals of 10 bases, demonstrating the precise control of the amino-modified density by Q5 polymerase PCR.

Referring to the method in example 1, two Linker NHS-DBCO and NHS-TCO were used to react with the DNA having amino group modification on the base prepared in example 1, and it is seen from FIG. 17A that the band after the reaction with NHS-DBCO is thinner than that with NHS-TCO, indicating that the reaction is more complete, so that NHS-DBCO is preferably used as Linker. FIG. 17B is a reaction of DBCO-ligated DNA with primers with azide modification at the 5-terminus.

Example 5 Fidelity detection of 5-Proparylamino-dCTP Using Q5 polymerase PCR

1. PCR was performed on 5-propargylamino-DNA. PCR system (final concentration): dATP, dGTP, dTTP,5-propargylamino-dCTP each 0.2mmol/L, Q5 polymerase 20U/ml,1 XQ 5 polymerase buffer, biotin-T1 (4 nmol/L), upstream primer T1-F (0.3. Mu. Mol/L, table 1), downstream primer T1-R (0.3. Mu. Mol/L, table 1). PCR was performed using the system (reaction conditions: 2min at 98 ℃, 10s at 98 ℃, 30s at 70 ℃, 1min at 72 ℃, 20 cycles; 5min at 72 ℃). The PCR product (5-propargylamino-DNA) was purified and then detected by 6% polyacrylamide gel electrophoresis and stained with Cyber gold.

2. PCR fidelity detection of 5-propargylamino-DNA

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

(2) The purified 5-propargylamino-DNA product was PCR amplified using dNTPs, the template for the PCR system was purified 5-propargylamino-DNA product, dNTPs 0.2mmol/L, Q5 polymerase 20U/mL,1 XQ 5 polymerase buffer, upstream primer T1-CL-F (0.5. Mu. Mol/L, table 1), and downstream primer T1-CL-R (0.5. Mu. Mol/L, table 1).

(3) Clone sequencing: the PCR product was digested with EcoRI and HindIII and inserted into the pUC19 plasmid digested with the same enzymes. The enzyme-linked product was chemically transformed into E.coli XL1-Blue cells and inoculated on ampicillin-containing agar plates at 37℃overnight. And selecting a single colony for PCR, sending positive clones to sequencing, comparing the sequencing result with the original T1 template, and observing whether mutation exists. As a result, as shown in FIG. 18, there were no mutations in the middle 29 bases (boxes) of 9 samples in total.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

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aactatat 1028

Claims

1. A preparation method of non-natural nucleic acid hydrogel is characterized by comprising the following steps: the method comprises the following steps:

(1) Preparation of upstream and downstream "brush" primers:

a. incorporating 5-propargylamine-modified artificial nucleotide 5-procyanidins-dCTP which can be paired and contains propargylamine at the 5 th position of the base part into a polymerase chain reaction system to prepare a nucleic acid fragment containing an amino connecting arm on part of the base; the polymerase in the polymerase chain reaction system is any one of Taq DNA polymerase, oneTaq DNA polymerase, Q5 DNA polymerase or SFM 4-3;

c. designing and synthesizing an upstream primer and a downstream primer according to the sequence of the target fragment, and modifying the 5' ends of the upstream primer and the downstream primer to form an azide group which can perform click chemical reaction with a linker;

d. C, carrying out click chemistry reaction on the nucleic acid fragment containing the linker on part of the nucleotide obtained in the step b and the upper and lower primers containing the modified azide group at the 5' end obtained in the step c respectively to obtain upper and lower hairbrush primers;

(2) Preparation of non-natural nucleic acid hydrogels:

performing PCR amplification by using a nucleic acid fragment or plasmid containing a target fragment as a template through the upstream and downstream brush primers obtained in the step (1), concentrating an amplification product, and annealing at a high temperature to obtain the non-natural nucleic acid hydrogel; the PCR amplification system contains 0.8-1.2 mol/L NaCl and 0.04-0.06% Tween20.

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

when the polymerase is SFM4-3, a pairing-able artificial nucleotide containing propargylamine group at the 5 th position of a base part is doped in a polymerase chain reaction system, a pairing-able artificial nucleotide containing fluorine group modification at the 2 nd position of a glycosyl part is doped in the polymerase chain reaction system, and a nucleic acid fragment containing the amino modification on the base and the fluorine group modification at the glycosyl is prepared, and then subsequent reaction is carried out;

and/or, the non-natural base dTTT 3 and dNaM are mixed in a polymerase chain reaction system to prepare a nucleic acid fragment containing the non-natural base pairs, and then subsequent reactions are carried out;

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

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

the click chemistry linker described in step b is selected from the group consisting of NHS-DBCO linker or NHS-TCO linker.

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

the manner of incorporation described in step a is to replace one or more of the natural nucleotides completely or partially;

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

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

the 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 described in step b was performed in a PBS buffer system with ph=8.4 to 8.6.

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

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

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

the reaction described in step d is carried out in a PBS buffer system having a ph=7.3 to 7.5.

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

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

the high temperature annealing operation in the step (2) is as follows: after heating at 95℃for 5min, it was gradually cooled to room temperature.

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

9. Use of the non-natural nucleic acid hydrogel of claim 8 in the preparation of a pharmaceutical carrier.