CN111040198A

CN111040198A - DNA hydrogel, preparation method and application thereof

Info

Publication number: CN111040198A
Application number: CN201911394424.XA
Authority: CN
Inventors: 杨修竹; 王丹丹; 贺亮; 庄淼; 崔金辉; 甘明哲
Original assignee: Suzhou Perotin Biotechnology Co Ltd
Current assignee: Suzhou Perotin Biotechnology Co Ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-04-21
Anticipated expiration: 2039-12-30
Also published as: CN111040198B

Abstract

The application discloses a DNA hydrogel, a preparation method thereof and application thereof in protein synthesis. The DNA hydrogel comprises linear DNA of modified functional groups and high molecular compounds containing the functional groups, which are formed by mutual crosslinking, can be applied to a cell-free protein synthesis system to obviously improve the protein expression amount and can be repeatedly used, and the cost for synthesizing the protein is effectively reduced.

Description

DNA hydrogel, preparation method and application thereof

Technical Field

The application relates to the field of hydrogel, in particular to DNA hydrogel, and a preparation method and application thereof.

Background

The protein has important scientific and practical significance as a potential therapeutic biological agent, a drug target and a biocatalyst. Due to the complexity of proteins, there is an increasing need to prepare and evaluate them in a simple manner. At present, most of the existing protein preparation methods are synthesized in living cells by using cells as hosts, but have many disadvantages: complicated operation, long period, extremely limited types of synthesized proteins and the like. The Cell-free Protein Synthesis (CFPS) system is an in vitro gene expression system which takes exogenous DNA or mRNA as a template, manually adds required raw materials and energy substances, and synthesizes Protein by taking Cell extracts as conditions, can break through Cell restriction and conveniently and quickly express various proteins. In detail, CFPS uses DNA as a template, and transcribes corresponding mRNA under the action of RNA polymerase, transcription factor and other components; the mRNA is used as a template, and ribosome, regulatory factor, amino acid substrate, tRNA, energy substance and the like in the system are utilized to translate to obtain synthetic protein. The unique advantage of CFPS over in vivo systems is the ability to directly utilize linear DNA templates (LETs) that can be amplified by Polymerase Chain Reaction (PCR), and there is no need to construct the target gene onto an expression vector, which significantly simplifies the work flow of CFPS and shortens the protein synthesis time.

CFPS based on LETs, which are effective tools for high throughput production and protein screening, have begun to demonstrate usefulness in various fields, such as: protein microarrays, cloned functional genomics, and the like. But due to the increased susceptibility of LETs to native nucleases in a cell-free environment compared to plasmids, it has a shorter lifetime and lower reaction yields.

To increase the lifetime of LETs in CFPS and the reaction yield of proteins, the methods adopted in the prior art may comprise, for example: (1) inhibit or reduce the activity of nuclease, protect linear DNA from degradation of nuclease, and increase the local concentration of template. (2) The preparation of cell extracts using strains in which the gene encoding endonuclease E (RNase E) has been knocked out delays the degradation of mRNA molecules and the level of proteins expressed from the LETs obtained by PCR amplification becomes comparable to that of conventional plasmid-based reactions, but has a problem in that the strain grows slowly and takes time. (3) The linear DNA fragments are formed into circular DNA molecules similar to plasmids by designing special primers, so that the resistance of the target gene fragments to exonuclease is improved, but the cost is high. (4) Applying the DNA hydrogel system to CFPS, primarily by ligating linear template DNA molecules with X-shaped DNA (X-DNA) to produce a DNA hydrogel for a combined cell-free transcription and translation system; in wheat germ cell-free systems, this method results in a 300-fold increase in protein production compared to soluble DNA templates, however X-DNA synthesis is expensive.

Disclosure of Invention

Aiming at the defects of the prior art, the following technical scheme is adopted in the application:

in a first aspect of the present application, a DNA hydrogel is provided, which is prepared from a linear DNA modified with a functional group and a high molecular compound containing a functional group.

Preferably, in the linear DNA for modifying the functional group, the functional group is selected from any one or more of an acrylamide group, a sulfhydryl group, an amino group or an azide group.

Preferably, in the functionalized group-containing polymer compound, the functionalized group may be selected from any one or more of an acrylate bond, a succinamide bond, an acrylamide bond, an amino group, or a thiol group.

Preferably, the functional group-containing polymer compound may be one or more of polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, or O, O' -bis [2- (N-succinimide-succinamido) ethyl ] polyethylene glycol.

Preferably, the number average molecular weight of the polyethylene glycol diacrylate is 250-4000 g/mol.

Preferably, the number average molecular weight of the O, O' -bis [2- (N-succinimide-succinamido) ethyl ] polyethylene glycol is 500-3000 g/mol.

A second aspect of the present application provides a method for preparing a DNA hydrogel, comprising:

providing linear DNA modifying a functional group;

providing a polymer compound containing a functional group; and

and mixing the high molecular compound containing the functional group with the linear DNA for modifying the functional group to perform a crosslinking reaction to obtain the DNA hydrogel.

Preferably, the crosslinking reaction comprises an addition reaction.

Preferably, in the linear DNA of the modified functional group, the modified functional group is selected from any one or more of an acrylamide group, a sulfhydryl group, an amino group or an azide group.

Preferably, in the functionalized group-containing polymer compound, the functionalized group may be selected from at least one or more of an acrylate bond, a succinamide bond, an acrylamide bond, an amino group, or a thiol group.

Preferably, the crosslinking reaction is carried out under the action of electromagnetic wave irradiation or a chemical crosslinking agent.

Preferably, the electromagnetic waves comprise any one or combination of ultraviolet light, visible light, near infrared light, or microwaves.

Preferably, the preparation method further comprises adding a free radical initiator to the crosslinking reaction.

Preferably, the free radical initiator comprises a persulfate, a peroxide or an azo compound.

Preferably, the preparation method further comprises adding a coagulant to the crosslinking reaction.

Preferably, the coagulant comprises tetramethylethylenediamine.

Preferably, the time required by the crosslinking reaction is 0.0001-24 h, and more preferably, the reaction time is 0.5-12 h.

Preferably, the crosslinking reaction temperature is 10-80 ℃, and more preferably, the reaction temperature is 25-60 ℃.

A third aspect of the present application provides a method of synthesizing a protein, comprising:

providing the DNA hydrogel or the DNA hydrogel prepared by the preparation method; and

synthesizing a protein from the DNA hydrogel.

Preferably, the method for synthesizing a protein is performed in the presence of a cell extract and an energy buffer.

Preferably, the energy buffer solution comprises an amino acid mixed solution, a reaction buffer solution and an energy supplementing solution.

Preferably, the cell extract is selected from at least one or more of escherichia coli cell extract, yeast cell extract, wheat germ extract, insect cell extract, rabbit reticulocyte extract and CHO cell extract.

Preferably, the amino acid mixture is an equimolar mixture prepared by adding deionized water to amino acid selected from the group consisting of alanine, arginine, serine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, lysine, phenylalanine, proline, tryptophan, threonine, valine, tyrosine, leucine, hemisarcosine, methionine, and asparagine, and the molar concentration of each amino acid is preferably 1 to 4 mmol/L.

Preferably, the reaction buffer solution comprises 40-330 mmol/L potassium glutamate, 3-6 mmol/L magnesium glutamate, 1-3% polyethylene glycol 8000, 1-3 mmol/L dithiothreitol and 14-18 mmol/L maltose, and the solvent is deionized water.

Preferably, the energy supplementing liquid comprises 50-60 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid (PH 8.0), 1-2 mmol/L adenosine triphosphate, 0.8-1 mmol/L uridine triphosphate, 0.8-1 mmol/L guanine-5 ' -triphosphate, 0.8-1 mmol/L cytidine triphosphate, 0.1-0.2 mg/L transport ribonucleic acid, 0.2-0.3 mmol/L coenzyme A, 0.3-0.4 mmol/L nicotinamide adenine dinucleotide, 0.6-0.9 mmol/L adenosine-3 ',5' -cyclic monophosphate, 0.03-0.07 mmol/L folinic acid, 10-40 mmol/L3-phosphoglyceride, 0.4-1 mmol/L spermidine, and the solvent is deionized water.

Preferably, the volume ratio of the cell extract to the energy buffer to the DNA hydrogel is (20-50): (10-60): (1-70).

The embodiment of the application adopts at least one technical scheme which can achieve the following beneficial effects:

1. the preparation method of the DNA hydrogel is simple and easy to operate and high in feasibility of implementation.

2. The DNA hydrogel obtained by protecting the linear DNA serving as the template is applied to a cell-free protein synthesis (CFPS) system, and can obviously improve the protein expression amount and the synthesis efficiency.

3. The DNA hydrogels of the present application can be reused in CFPS systems, reducing the cost required to synthesize proteins using linear DNA templates.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is a flow chart of a method for preparing a DNA hydrogel according to the present application;

FIG. 2 is a fluorescent image of the DNA hydrogel of example 1 of the present application after staining with a nucleic acid dye;

FIG. 3 is a scanning electron microscope image of DNA hydrogels containing different concentrations of PEGDA of example 1 of the present application;

FIG. 4 is a graph showing the protein expression levels of the DNA hydrogel and the liquid phase system of example 1 of the present application;

FIG. 5 is a graph showing the effect of PEGDA concentration on the expression level of a fluorescent protein in example 1 of the present application;

FIG. 6 is a graph showing the effect of APS concentration on the expression level of fluorescent protein in example 1 of the present application;

FIG. 7 is a graph showing the effect of cross-linking time on the expression level of fluorescent protein in example 1 of the present application;

FIG. 8 is a graph showing the effect of the amount of DNA in a DNA hydrogel on the expression level of cell-free protein in example 1 of the present application;

FIG. 9 is a histogram of protein expression levels obtained by repeated use of the DNA hydrogel of example 1 of the present application;

FIG. 10 is a graph of protein expression levels of a DNA hydrogel and a liquid phase system according to example 2 of the present application;

FIG. 11 shows the effect of single-and double-ended modifications of DNA templates on cell-free protein expression of DNA hydrogels in example 3 of the present application;

FIG. 12 is a histogram showing the protein expression levels obtained by repeating the use of the DNA template single-end modified DNA hydrogel in example 3 of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the following specific embodiments of the present application and the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the present application and not restrictive of the total embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the event that a definition used herein conflicts or disagrees with a definition contained in another publication, the definition used herein shall govern.

As used herein, the terms "selected from", "consisting of …" and "consisting of" are synonymous with "comprising". As used herein, the terms "comprises," "comprising," "includes," "including," "has," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a plurality of elements listed in a list is not necessarily limited to only those elements listed in the list, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range is recited as "1 to 5", the recited range should be understood to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and so forth. If a range of values is recited in this specification, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range.

In addition, unless expressly stated otherwise, "or" means an inclusive "or" and not an exclusive "or. For example, condition A "" or "" B satisfies any of the following conditions: a is true (or present) and B is false (or not present), a is false (or not present) and B is true (or present), and both a and B are true (or present).

Similarly, the indefinite articles "a" and "an" preceding an element or component herein are intended to describe without limitation the number of instances (i.e., occurrences) of the element or component, unless the context clearly indicates otherwise. Thus, "a", "an", and "an" should be understood to include one or at least one, and the singular forms of the elements or components also include the plural.

Several terms herein are defined as follows:

the term "Polymerase Chain Reaction (PCR)" refers to a method for amplifying a specific nucleotide sequence (amplimer). PCR relies on the activity of a nucleic acid polymerase to extend primers on a template to obtain an amplicon. Preferably, the nucleic acid polymerase is thermostable.

The term "expression" refers to the process of transcription of DNA into mRNA and/or the further translation of the transcribed mRNA into a peptide, polypeptide or protein.

FIG. 1 is a flow chart of a method of preparing a DNA hydrogel of the present application, which may include the steps of:

step S11 is to provide linear DNA that modifies the functional group. The functional group is, for example, an acrylamide group, a thiol group, an amino group, or an azide group, and can be modified at the 3 'end and/or the 5' end of the linear DNA. For example, linear DNA modified with a functional group can be prepared by modifying the functional group to the 5' end of a primer and performing PCR amplification. The linear DNA modifying the functional group may be, for example, Acrydite-DNA, HS-DNA, H₂N-DNA、N₃DNA and the like.

Step S12 is to provide a polymer compound containing a functionalized group. The functional groups contained in the macromolecular compound can be selected from an acrylic ester bond, a succinamide bond, an acrylamide bond or a sulfydryl, wherein the functional groups at least comprise two groups; preferably, both ends of the polymer compound are modified by functional groups.

The polymer compound containing a functional group may be polyethylene glycol diacrylate (PEGDA), O '-Bis [2- (N-succinimide-succinylamino) ethyl ] polyethylene glycol (O, O' -Bis [2- (N-succinimide-ethyl ] polyethylene glycol), etc.

Step S13 is to perform cross-linking reaction between the polymer compound containing the functional group and the linear DNA of the modified functional group to obtain the DNA hydrogel. The crosslinking reaction may include an addition reaction. Preferably, the crosslinking reaction can be carried out under the action of electromagnetic wave irradiation or the addition of a chemical crosslinking agent. The electromagnetic wave may be ultraviolet light, visible light, near infrared light, microwave, or a combination thereof. The chemical crosslinking agent may for example be a persulfide, a peroxide, an azo compound. The prepared DNA hydrogel has porosity and is applied to a cell-free protein synthesis (CFPS) system.

Preferably, a radical initiator may also be added to the crosslinking reaction, for example: persulfides, peroxides, and azo compounds. For example, the persulfides may be potassium persulfate (KPS), sodium persulfate (NaPS), and Ammonium Persulfate (APS), with ammonium persulfate and potassium persulfate being the most commonly used. The most commonly used peroxides are Benzoyl Peroxide (BPO), and others are bis (2,4-dichlorobenzoyl) peroxide, diacetyl peroxide, dioctanoyl peroxide and dilauroyl peroxide (LPO). Azo compounds are commonly used, including Azobisisobutyronitrile (AIBN) and Azobisisoheptonitrile (ABVN). In the crosslinking reaction, a coagulant can be further added to improve the reaction efficiency. The coagulant may be Tetramethylethylenediamine (TEMED).

The DNA hydrogel can be applied to cell-free protein production, and not only can protect a PCR product serving as a template, but also can increase the local effective concentration of the template. Therefore, the DNA hydrogel can be used as a template in a CFPS system repeatedly for a plurality of times, so that the reaction cost can be greatly reduced.

In one embodiment of the present application, PEGDA is selected as the polymer compound, which has hydrophilicity and good biocompatibility, and does not inhibit protein synthesis in CFPS system.

(1) Preparation of DNA hydrogel using PEGDA:

PEGDA (number average molecular weight: 250-4000 g/mol, final volume concentration: 1-15%) and 10-1000 ng/. mu.L of linear DNA of a modifying functional group are added into a buffer solution (such as PBS (pH 7.4), TBE (pH 8) or HEPES-KOH (pH 8)), mixed uniformly, and then the mixture is kept stand at 25-60 ℃ for reaction for 0.5-12 hours, thus obtaining the DNA hydrogel. In another embodiment, 0.5-8 mg/mL APS can be added as a free radical initiator to accelerate the reaction and/or improve the reaction efficiency.

In another embodiment, TEMED can be added as a coagulant at a concentration of 0.05% to 5% to accelerate the reaction and/or improve the reaction efficiency.

In another embodiment, 0.5-8 mg/mL APS and TEMED (concentration of 0.05% -5%) can be added simultaneously to prepare the DNA hydrogel so as to accelerate the reaction and/or improve the reaction efficiency.

(2) DNA hydrogels prepared with PEGDA were applied to cell-free protein synthesis (CFPS) system:

mixing (I) a cell extract, (II) an energy buffer solution and (III) a DNA hydrogel prepared by PEGDA in any one of the above embodiments in a volume ratio of (20-50): (10-60): (1-70), placing the mixture into a constant-temperature reaction container at the temperature of 4-37 ℃, and shaking the mixture at the rotating speed of 300-1500 rpm for 0.5-24 hours to obtain a solution containing the expression protein.

In another embodiment of the present application, O' -bis [2- (N-succinimidyl-succinamido) ethyl ] polyethylene glycol is used as the functionalized polymeric compound.

(1) Preparation of DNA hydrogel Using O, O' -bis [2- (N-succinimidyl-succinamido) ethyl ] polyethylene glycol:

adding O, O' -bis [2- (N-succinimidyl-succinamido) ethyl ] polyethylene glycol (number average molecular weight: 500-3000 g/mol, final volume concentration: 1-15%) and linear DNA of a modified functional group into a buffer (such as PBS (PH 7.4), TBE (PH 8) or HEPES-KOH (PH 8)), mixing uniformly, standing at 10-80 ℃, and reacting for 0.5-12 hours to obtain the DNA hydrogel.

In another embodiment, 0.5-8 mg/ml of LAPS can be added as a free radical initiator to accelerate the reaction and/or improve the reaction efficiency.

In another embodiment, 0.5-8 mg/mL APS and TEMED (concentration of 0.05% -5%) can be added simultaneously to prepare the DNA hydrogel so as to accelerate the reaction and/or improve the reaction efficiency. (2) The DNA hydrogel prepared from O, O' -bis [2- (N-succinimide-succinamido) ethyl ] polyethylene glycol is applied to a CFPS system:

preparing a DNA hydrogel from (I) a cell extract, (II) an energy buffer solution and (III) O, O' -bis [2- (N-succinimide-succinamido) ethyl ] polyethylene glycol in any one of the above embodiments, wherein the volume ratio of the DNA hydrogel is (20-50): (10-60): (1-70), placing the mixture into a constant-temperature reaction container at the temperature of 4-37 ℃, and shaking the mixture at the rotating speed of 300-1500 rpm for 0.5-24 hours to obtain a solution containing the expression protein.

In both embodiments, the cell extract source includes eukaryotic cells or prokaryotic cells, and the CFPS system preferably includes, but is not limited to, escherichia coli cell-free system (ECE. coli S30 extract), Wheat germ cell-free system (WGE), Rabbit reticulocyte cell-free system (RRL), or any combination thereof. The energy buffer solution comprises an amino acid mixed solution, a reaction buffer solution and an energy supplementing solution.

The specific embodiment is as follows:

example 1

(1) Preparation of Acrydite-DNA template:

pIJ8660-GFP plasmid is obtained by DNA complete sequence synthesis, PCR primers are designed according to GFP gene, a forward primer sfGFP-FOR sequence is shown as SEQ ID No.1, a reverse primer sfGFP-REV sequence is shown as SEQ ID No.2, a functional group 6-methacrylamidohexylphosphate (Acrydite) is modified at the 5' end of the primer (Table 1), and the modified primers and the pIJ8660-GFP plasmid are mixed FOR PCR reaction to obtain a PCR product Acrydite-DNA of the target gene GFP, which is used as a template FOR cell-free protein synthesis (CFPS) of the embodiment.

TABLE 1

(2) Preparation of DNA hydrogel:

mu.L of PEGDA (number average molecular weight: 575) or 0.5. mu.L of PEGDA (number average molecular weight: 575), 5.7. mu.L of LAcrydite-DNA (concentration: 185 ng/. mu.L), 2. mu.L of LAPS (concentration: 0.01mg/ml) and 1% TEMED were added to HEPES-KOH buffer at pH 8, and the mixture was mixed uniformly in a total volume of 10. mu.L, allowed to stand and subjected to a crosslinking reaction for 5 hours, to thereby obtain DNA hydrogels containing 2% PEGDA and 5% PEGDA by volume, respectively. As shown in FIG. 2, the DNA hydrogel obtained in this example was stained with a nucleic acid dye, and a fluorescence image was taken, which confirmed that Acrydite-DNA was efficiently crosslinked in the hydrogel.

Referring to fig. 3, which is an image of the DNA hydrogel prepared in this example taken by a scanning electron microscope, it can be seen that the DNA hydrogel with 2% PEGDA by volume or the DNA hydrogel with 5% PEGDA by volume has a porous structure, and the adjustment of the void size of the DNA hydrogel can be achieved by adjusting the PEGDA concentration.

(3) Application of DNA hydrogel to CFPS system

Preparation of cell extract:

firstly, culturing an escherichia coli strain, then collecting thalli, and performing actions such as suspension, centrifugation, low-temperature high-pressure crushing, dialysis, liquid nitrogen cold shock and the like to obtain a cell crushed product, and subpackaging and storing the cell crushed product in an environment at-80 ℃.

Preparation of amino acid mixed solution:

the selected amino acid components are shown in the table below, wherein the used solvent is deionized water, and the following amino acid components are mixed with the deionized water and then are complemented to 10mL to obtain an amino acid mixed solution.

TABLE 2

Preparation of reaction buffer:

the components of the selected reaction buffer are shown in the table below, wherein the used solvent is deionized water, and the following components are mixed with the deionized water and then are made up to 10mL to obtain the reaction buffer.

TABLE 3

Components of the reaction buffer	Adding amount of
		Potassium glutamate	100mmol/L
Glutamic acid magnesium salt	4mmol/L
		Polyethylene glycol 8000	2％
Dithiothreitol	2mmol/L
		Maltose	15mmol/L

Preparation of energy replenisher

The components of the selected energy supplementing liquid are shown in the table, wherein the used solvent is deionized water, and the following components are mixed with the deionized water and then are made up to 10mL to obtain the energy supplementing liquid.

TABLE 4

The overall component allocation for the cell-free protein synthesis (CFPS) system is shown in the following table:

TABLE 5

Components of CFPS systems	Adding amount of
		Cell extract	6.67μL
Amino acid mixed liquor	7.13μL
		Reaction buffer	1.77μL
Energy supplementing liquid	1.43μL
		DNA hydrogel	1.00μL(200ng/L)
Deionized water	2.00μL

In summary, the volume ratio of the cell extract, the energy buffer composed of the amino acid mixture, the reaction buffer and the energy supplement, and the DNA hydrogel prepared from PEGDA and Acrydite-DNA was 33.3: 51.7: 5, placing the mixture in a PCR tube, using an Instrument thermo mixer, shaking the mixture for 13 hours at 30 ℃ and 1000rpm, taking the mixture out, and carrying out real-time quantitative detection on the green fluorescence intensity of the mixture by an Enzyme-linked immunosorbent assay (Enzyme-labeled Instrument) to indirectly reflect the expression quantity of the green fluorescent protein by the relative intensity of fluorescence.

FIG. 4 is a graph showing the protein expression levels of the DNA hydrogel of example 1 and a liquid phase system (SPS). The liquid phase system is used as a comparative example, which is to directly perform a PCR reaction using unmodified primers to obtain a PCR product of the target gene GFP as a template of CFPS, i.e., the used template is only linear DNA. As shown in FIG. 4, the DNA hydrogels containing 2% PEGDA and 5% PEGDA prepared in this example were significantly improved in protein expression when applied to the synthesis of proteins, as compared to the linear PCR product used in the liquid phase system.

Effect of PEGDA concentration on fluorescent protein expression

As seen from fig. 5, when the concentration of linear DNA was constant, the fluorescence intensity increased as the concentration of PEGDA increased from 0.5% to 2%; the fluorescence intensity reached a maximum when the PEGDA concentration was 2%; as PEGDA concentration increased from 2% to 15%, fluorescence intensity decreased; the fluorescence intensity was not significantly different from the SPS system when the PEGDA concentration was 0.5% and 1%. The reason may be that when the PEGDA concentration is 0.5% and 1%, hydrogel cannot be formed, linear DNA cannot be protected, and fluorescence intensity does not increase. When the PEGDA is 2%, a hydrogel can be formed, and as the PEGDA concentration increases, the mechanical strength of the hydrogel increases, the pore size becomes smaller, the substance exchange rate becomes lower, and the fluorescence intensity decreases.

Effect of APS concentration on fluorescent protein expression

In chemical gel-forming systems, APS acts as an initiator, and the higher the concentration, the lower the fluorescence intensity of the CFPS system. As seen from FIG. 6, the fluorescence intensity was maximized at the APS final concentration of 0.1% or 0.2%. The reason may be that the APS concentration is increased, which leads to the increase of free radicals in the gel system, which may damage the DNA, and the template DNA cannot be transcribed and translated, resulting in the decrease of fluorescence intensity.

Effect of Cross-linking time on fluorescent protein expression

As can be seen from FIG. 7, the cross-linking time required only 2h to form a stable hydrogel.

Effect of DNA concentration on fluorescent protein expression

As can be seen from FIG. 8, the fluorescence intensity increased with the increase in the concentration of template DNA, probably because the higher the concentration of DNA, the smaller the proportion of exonuclease-degraded DNA, and the higher the fluorescence intensity of the CFPS system. However, when the amount of DNA in the hydrogel was 300ng, the fluorescence intensity reached a plateau.

Reuse of DNA hydrogels

This example was conducted to confirm whether the DNA hydrogel prepared in the present application has reusable characteristics, in a manner similar to that in the CFPS system, using the method and conditions as described in example 1 above, the cell extract, energy buffer, and DNA hydrogel as a template were added to complete the expression of fluorescent protein and quantitatively detect the green fluorescence intensity thereof, and then fresh cell extract was supplemented to reuse the hydrogel, and repeated 10 times, and the fluorescence intensity measured each time was recorded one by one, and the experimental results are shown in FIG. 9. As can be seen from the histogram shown in FIG. 9, the DNA hydrogel of the present application enables synthesis of a protein in high yield even when it has been repeatedly used 10 times.

Specific example 2:cell-free protein expression of DNA hydrogels

(1) Preparation of HS-DNA template:

pIJ8660-GFP plasmid is obtained by DNA complete sequence synthesis, PCR primers are designed according to GFP gene, a forward primer sfGFP-FOR sequence is shown as SEQ ID No.1, a reverse primer sfGFP-REV sequence is shown as SEQ ID No.2, Thiol-Modifier C6S-S (table 2) of a functional group sulfhydryl is modified at the 5' end of the primer, and the modified primer is mixed with the pIJ8660-GFP plasmid FOR PCR reaction to obtain a PCR product HS-DNA of the target gene GFP, which is used as a template FOR cell-free protein synthesis (CFPS) of the embodiment.

TABLE 2

(2) Preparation of DNA hydrogel:

mu.L of PEGDA (number average molecular weight: 575) or 0.5. mu.L of PEGDA (number average molecular weight: 575), 5.7. mu.L of LHS-DNA (concentration: 185 ng/. mu.L), 2. mu.L of LAPS (concentration: 0.01mg/ml), and 1% TEMED were added to HEPES-KOH buffer solution at pH 8, and the mixture was mixed well in total volume of 10. mu.L, allowed to stand, and subjected to a crosslinking reaction for 5 hours, to thereby obtain DNA hydrogels containing 2% PEGDA and 5% PEGDA by volume, respectively.

(3) Application of DNA hydrogel to CFPS system

The DNA hydrogel prepared from the cell extract, the energy buffer composed of the amino acid mixture, the reaction buffer, and the energy supplement solution, and the PEGDA and HS-DNA according to the method of example 1, was mixed at a volume ratio of 33.3: 51.7: 5, placing the mixture in a PCR tube, using an Instrument thermo mixer, shaking the mixture for 13 hours at 30 ℃ and 1000rpm, taking the mixture out, and carrying out real-time quantitative detection on the green fluorescence intensity of the mixture by an Enzyme-linked immunosorbent assay (Enzyme-labeled Instrument) to indirectly reflect the expression quantity of the green fluorescent protein by the relative intensity of fluorescence.

FIG. 10 is a graph showing the protein expression levels of the DNA hydrogel of example 2 and a liquid phase system (SPS). The liquid phase system is used as a comparative example, which is to directly perform a PCR reaction using unmodified primers to obtain a PCR product of the target gene GFP as a template of CFPS, i.e., the used template is only linear DNA. As shown in FIG. 10, the DNA hydrogels containing 2% PEGDA and 5% PEGDA prepared in this example were significantly improved in protein expression when applied to the synthesis of proteins, as compared to the linear PCR product used in the liquid phase system.

Specific example 3: effect of Single-and double-ended modification of DNA templates on cell-free protein expression of DNA hydrogels

(1) Single-ended Acrydite-DNA template

pIJ8660-GFP plasmid is obtained by DNA full sequence synthesis, a PCR primer is designed according to GFP gene, a forward primer sfGFP-FOR sequence is shown as SEQ ID No.1, a reverse primer sfGFP-REV sequence is shown as SEQ ID No.2, a functional group is modified at the 5' end of the forward primer, 6-methacrylamidohexylphosphate (Acrydite) with an acrylamide group is grafted, and then the modified forward primer and an unmodified reverse primer (Table 3) are used FOR obtaining the Acrydite-DNA modified by the single-ended product of the target gene GFP and used as a template FOR cell-free protein synthesis (CFPS) of the embodiment.

TABLE 3

(2) The double-ended Acrydite-DNA template preparation procedure was as described in example 1.

(3) The DNA hydrogel was prepared by adding 0.2. mu.L of PEGDA (number average molecular weight: 575), 5.7. mu.L of LAcrydite-DNA (concentration: 185 ng/. mu.L), 2. mu.L of LAPS (concentration: 0.01mg/ml) and 1% TEMED to HEPES-KOH, which was a buffer solution having pH 8, in a total volume of 10. mu.L, mixing them uniformly, and allowing to stand for a crosslinking reaction for 5 hours, as described in example 1.

(4) The cell-free protein expression method was as described in example 1, and the results are shown in FIG. 11. The liquid phase system is used as a comparative example, which is to directly perform a PCR reaction using unmodified primers to obtain a PCR product of the target gene GFP as a template of CFPS, i.e., the used template is only linear DNA. Compared with the linear PCR product used in the liquid phase system, the DNA hydrogel with single-end modification and double-end modification of the DNA template prepared in this example can significantly improve the protein expression level when the DNA hydrogel is applied to the synthesis of protein.

(5) The DNA hydrogel was reused as described in example 1, and the results are shown in FIG. 12. The DNA hydrogel with the single-end modified DNA template prepared in the embodiment can be reused only a few times, and the DNA hydrogel with the double-end modified DNA template can still synthesize and obtain high-yield protein even if the DNA hydrogel is reused for 10 times. Therefore, the stability of the DNA hydrogel with the double-end modified DNA template is far higher than that of the DNA hydrogel with the single-end modified DNA template.

In summary, the DNA hydrogel of the present application can protect a linear DNA template, and when applied to a CFPS system, can significantly increase the amount of protein expression. Furthermore, the DNA hydrogel of the present application can be reused, so that the amount of template for protein synthesis by PCR amplification can be reduced, and the cost required for protein synthesis can be effectively saved.

The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Comparative example 1: protein expression of linear DNA templates in cell-free energy systems

1) Preparation of cell extracts as described in example 1;

2) formulation of an energy replenishment system, as described in example 1;

3) protein expression of linear DNA template in cell-free energy system:

components of CFPS systems	Adding amount of
		Cell extract	6.67μL
Amino acid mixed liquor	7.13μL
		Reaction buffer	1.77μL
Energy supplementing liquid	1.43μL
		Linear DNA	1.00μL(200ng/L)
Deionized water	2.00μL

The cell extract, energy buffer solution consisting of amino acid mixed solution, reaction buffer solution and energy supplement solution and linear DNA are uniformly mixed and placed in a PCR tube, an Instrument ThermoMixer is used, the cell extract is taken out after being shaken for 13 hours under the conditions of 30 ℃ and 1000rpm, and an Enzyme-linked immunosorbent assay (Enzyme-labeled Instrument) is used for carrying out real-time quantitative detection on the green fluorescence intensity of the cell extract, so that the expression quantity of the green fluorescent protein is indirectly reflected by the relative intensity of fluorescence.

Sequence listing

<110> Perot biotech, Suzhou Ltd

<120> DNA hydrogel, preparation method and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>1

tggagcggat cggggattgt 20

<210>2

<211>20

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400>2

ccggtcgact ctagctagag 20

Claims

1. The DNA hydrogel is characterized in that raw materials for preparing the DNA hydrogel comprise linear DNA for modifying functional groups and macromolecular compounds containing the functional groups.

2. The DNA hydrogel of claim 1, wherein the linear DNA modifying functional group is selected from the group consisting of acrylamide, thiol, amino and azide groups.

3. The DNA hydrogel according to claim 1, wherein the functional group-containing polymer compound comprises a functional group selected from at least one or more of an acrylate bond, a succinamide bond, an acrylamide bond, an amino group and a thiol group.

4. The DNA hydrogel of claim 3, wherein the functionalized polymer compound is selected from the group consisting of polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and O, O' -bis [2- (N-succinimidyl-succinamido) ethyl ] polyethylene glycol.

5. The DNA hydrogel according to claim 4, wherein the number average molecular weight of the polyethylene glycol diacrylate is 250 to 4000 g/mol.

6. The DNA hydrogel according to claim 4, wherein the number average molecular weight of the O, O' -bis [2- (N-succinimide-succinamido) ethyl ] polyethylene glycol is 500 to 3000 g/mol.

7. A method for preparing a DNA hydrogel, comprising:

providing linear DNA modifying a functional group;

providing a polymer compound containing a functional group; and

8. The method of claim 7, wherein the crosslinking reaction comprises an addition reaction.

9. The method according to claim 7, wherein the linear DNA modifying the functional group has a functional group selected from the group consisting of an acrylamide group, a thiol group, an amino group and an azide group.

10. The method according to claim 7, wherein the functional group-containing polymer compound is a compound in which a functional group is selected from at least one or more of an acrylate bond, a succinamide bond, an acrylamide bond, an amino group, and a thiol group.

11. The method according to claim 10, wherein the polymer compound having a functional group is selected from the group consisting of polyethylene glycol diacrylate, polyethylene glycol dimethacrylate and O, O' -bis [2- (N-succinimide-succinamido) ethyl ] polyethylene glycol.

12. The method according to claim 7, wherein the crosslinking reaction is carried out by irradiation of electromagnetic waves or a chemical crosslinking agent.

13. The method according to claim 12, wherein the electromagnetic wave comprises any one or a combination of ultraviolet light, visible light, near-infrared light, or microwaves.

14. The method of claim 7, further comprising adding a free radical initiator to the crosslinking reaction.

15. The method of claim 14, wherein the free radical initiator comprises any one or more of a persulfate, a peroxide, or an azo compound in combination.

16. The method of claim 7, further comprising adding a coagulant to the crosslinking reaction.

17. The method of claim 16, wherein the coagulant comprises tetramethylethylenediamine.

18. The method according to claim 7, wherein the time required for the crosslinking reaction is 0.0001 to 24 hours.

19. The method according to claim 7, wherein the crosslinking reaction temperature is 10 to 80 ℃.

20. A method of synthesizing a protein, comprising:

providing the DNA hydrogel according to any one of claims 1 to 6, or the DNA hydrogel prepared by the preparation method according to any one of claims 7 to 17; and synthesizing a protein through the DNA hydrogel.

21. The method of claim 20, wherein the protein synthesis is performed in the presence of a cell extract and an energy buffer.

22. The method of synthesizing a protein according to claim 21, wherein the energy buffer comprises an amino acid mixture, a reaction buffer, and an energy supplement.

23. The method for synthesizing protein according to claim 21, wherein the cell extract is selected from at least one or more of escherichia coli cell extract, yeast cell extract, wheat germ extract, insect cell extract, rabbit reticulocyte extract, and CHO cell extract.

24. The method for synthesizing protein according to claim 22, wherein the amino acid mixture is a mixture of at least one or more of twenty amino acids selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.

25. The method of claim 22, wherein the reaction buffer comprises 40-330 mmol/L potassium glutamate, 3-6 mmol/L magnesium glutamate, 1-3% polyethylene glycol 8000, 1-3 mmol/L dithiothreitol, and 14-18 mmol/L maltose, and the solvent is deionized water.

26. The method for synthesizing a protein according to claim 22, wherein the energy-supplementing solution comprises 50 to 60 mmol/L4-hydroxyethylpiperazine ethanesulfonic acid, 1 to 2mmol/L adenine nucleoside triphosphate, 0.8 to 1mmol/L uridine triphosphate, 0.8 to 1mmol/L guanine-5 ' -triphosphate, 0.8 to 1mmol/L cytidine triphosphate, 0.1 to 0.2mg/mL transport ribonucleic acid, 0.2 to 0.3mmol/L coenzyme A, 0.3 to 0.4mmol/L nicotinamide adenine dinucleotide, 0.6 to 0.9mmol/L adenosine-3 ',5' -cyclic monophosphate, 0.03 to 0.07mmol/L folinic acid, 10 to 40 mmol/L3-phosphoglyceride, 0.4 to 1mmol/L spermidine, and the solvent is deionized water.

27. The method for synthesizing protein according to claim 21, wherein the volume ratio of the cell extract, the energy buffer solution and the DNA hydrogel is (20-50): (10-60): (1-70).