CN112661987B - Functionalized DNA hydrogel, preparation method and application thereof - Google Patents

Abstract

The application discloses a functionalized DNA hydrogel, a preparation method and application thereof. The preparation method comprises the following steps: and (3) crosslinking a uniform mixed system containing the maleimide group-containing high molecular compound, the thiol group-containing high molecular compound, the dopamine and the thiol-modified DNA at room temperature to obtain the functional DNA hydrogel. The preparation process of the functional DNA hydrogel provided by the application is simple, quick and low in cost, does not need conditions such as initiation, coagulation acceleration and the like, has high gel forming speed in preparation, is generally within 60 seconds, has high feasibility, and can be used for cell-free protein expression; meanwhile, the functional DNA hydrogel disclosed by the application has higher mechanical strength and is not easy to break, so that not only can the PCR product modified by sulfhydryl groups be protected, but also the local effective concentration of a template can be increased, and the protein expression quantity is further improved; the hydrogel can be reused, so that the protein expression cost is reduced.

Description

Functionalized DNA hydrogel, preparation method and application thereof

Technical Field

The application relates to a novel hydrogel, in particular to a functionalized DNA hydrogel for cell-free protein expression (cell-free protein synthesis, CFPS) and a preparation method and application thereof, and belongs to the technical field of hydrogels.

Background

Currently, protein drugs such as antibodies, polypeptides, etc. are becoming more and more widespread in the treatment of diseases, but the problem faced at present is that antibody drugs such as targeted drugs for the treatment of cancers are expensive, and many patients often abandon the treatment because they are not affordable. The high cost of antibody-based drugs is mainly due to the high cost. At present, the main mode of protein medicine production is that living cells are produced and then extracted, the operation steps are more, cell culture is needed, and pollution is easy to be caused in the culture process, so that the production cost of the protein medicine is increased. The advent of cell-free protein synthesis (cell-free protein synthesis, CFPS) systems has provided a potential solution to the above-described problems. The cell-free protein synthesis is an in vitro life simulation system independent of the whole cell structure, uses protein factors, enzymes and protein synthesis machines in cell extracts, uses exogenous DNA or mRNA as templates, supplements amino acids, energy and the like, realizes in vitro expression of target proteins, provides a powerful platform for in vitro synthesis of proteins and peptides, and provides a non-biological approach for high-efficiency expression of proteins. The CFPS breaks through the physiological limit of cells and has the unique advantages that the CFPS can directly utilize a linear DNA template, such as a PCR product, for example, a plasmid containing a target gene is converted into the linear template through PCR, and the CFPS system is directly utilized for protein expression, so that the protein synthesis time is shortened. However, since linear templates are susceptible to degradation by nucleases in cell-free expression systems, the concentration of templates is reduced, thereby reducing the efficiency of protein expression.

At present, researchers in the industry mainly start to protect linear templates from two directions to improve protein expression efficiency in CFPS. One direction is to inhibit or reduce nuclease activity; the second direction is to protect the linear DNA from reduced contact with nucleases to reduce template degradation and increase the local concentration of template.

Ahn et al used the strain from which the gene encoding endonuclease E was knocked out to prepare cell-free extracts, delayed the degradation of the mRNA molecules and the level of protein expressed from the template obtained by PCR amplification became comparable to conventional plasmid-based reactions. However, there is a problem in that the strain grows slowly and has a long period.

Gams protein from phage lambda was identified as an effective inhibitor of RecBCD nuclease, a truncated form of Gams of this protein has been used for cell-free protein expression, nuclease activity was inhibited, and the protein expression amount could reach nearly plasmid level, but there was a problem in that it was necessary to synthesize and purify this protein in bacteria, and the operation was cumbersome.

The Luo group reports a DNA clay hybrid hydrogel that mimics the blocking function of cells by electrostatic interactions between DNA and clay, and clay hydrogels can provide an effective closed environment, protecting genes from nucleases. When they further used this mixed hydrogel in CFPS systems, the protein yield was 6 times higher than in liquid phase systems. However, the clay hydrogel has lower mechanical strength, is difficult to recover after expression, and brings difficulty to recycling.

In conclusion, in the prior art, the strain is directly subjected to genetic modification, so that the strain grows slowly and has a longer period; the mode of synthesizing the RecBCD nuclease inhibitor requires the synthesis and purification of protein, and has complex operation; the clay hydrogel is used, so that the mechanical strength is low, and the clay hydrogel is difficult to recycle.

Disclosure of Invention

The application mainly aims to provide a functionalized DNA hydrogel and a preparation method thereof, which are used for overcoming the defects in the prior art.

It is another object of the present application to provide the use of said functionalized DNA hydrogel in cellular protein expression.

In order to achieve the aim of the application, the application adopts the following technical scheme:

the embodiment of the application provides a functional DNA hydrogel which is prepared by a cross-linking reaction of a macromolecular compound containing maleimide groups, a macromolecular compound containing sulfhydryl groups, dopamine and DNA modified by sulfhydryl groups.

In some embodiments, the maleimide-containing polymeric compound or thiol-containing polymeric compound is multi-armed.

Further, the maleimide group-containing polymer compound includes a four-arm polyethylene glycol maleimide.

Further, the thiol-group-containing polymer compound comprises a four-arm polyethylene glycol thiol group.

The embodiment of the application also provides a preparation method of the functionalized DNA hydrogel, which comprises the following steps:

and (3) crosslinking a uniform mixed system containing the maleimide group-containing high molecular compound, the thiol group-containing high molecular compound, the dopamine and the thiol-modified DNA at room temperature to obtain the functionalized DNA hydrogel.

The embodiment of the application also provides the functionalized DNA hydrogel prepared by the method.

The embodiment of the application also provides application of the functionalized DNA hydrogel in the field of cell-free protein expression (cell-free protein synthesis, CFPS).

Accordingly, the embodiment of the application also provides a method for synthesizing protein, which comprises the following steps:

providing the functionalized DNA hydrogel described previously;

the functionalized DNA hydrogel is used for synthesizing protein.

In some embodiments, the method specifically comprises: the cell extract, the energy buffer solution and the hydrogel are mixed according to the volume ratio of (10-100): (10-100): (1-50), and shaking the obtained mixed solution for 0.5-48 hours at the temperature of 0-37 ℃ and the rotating speed of 0-2000rpm to obtain a solution containing expressed protein, and further synthesizing the protein.

Compared with the prior art, the application has the beneficial effects that:

1) The preparation process of the functional DNA hydrogel provided by the application is simple, quick and low in cost, does not need conditions such as initiation, coagulation acceleration and the like, has high gel forming speed in preparation, is generally within 60 seconds, has high feasibility, and can be used for cell-free protein expression;

2) The functional DNA hydrogel provided by the application has high mechanical strength, is not easy to break, can protect a template, and further improves the protein expression quantity; the hydrogel can be reused, so that the cost required by amplification products is reduced, and the protein expression cost is reduced;

3) The functional DNA hydrogel material provided by the application is applied to cell-free protein production, can protect a sulfhydryl modified PCR product, and can increase the local effective concentration of a template. The application realizes the improvement of the protein yield, the template can be reused for a plurality of times, and the reaction cost is reduced.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to the drawings without inventive effort to those skilled in the art.

FIGS. 1a and 1b are schematic illustrations of the reaction between a polymer and a modifying group on DNA to form a hydrogel in an exemplary embodiment of the application;

FIG. 2 is a graph showing the difference between the protein expression levels of the functionalized DNA hydrogel of example 1 of the present application and that of the liquid phase system;

FIG. 3 is a graphical representation of the results of repeated use of functionalized DNA hydrogels of test example 1 of the present application;

FIG. 4 is a graph showing the expression level of functionalized DNA hydrogel proteins at different concentrations in example 2 of the present application;

FIG. 5 is a graph showing the expression level of functionalized DNA hydrogel proteins formed from polymers of different molecular weights in example 2 of the present application;

FIG. 6 is a graph showing the effect of different pH values on the gel formation time of the reaction between the thiol group of the four-arm polyethylene glycol and the maleimide group of the four-arm polyethylene glycol in example 2 of the present application;

FIG. 7 is a schematic diagram showing the relationship between the elastic modulus (G ') and the viscous modulus (G') of the functionalized DNA hydrogel of test example 2 of the present application.

Detailed Description

As described above, in view of the drawbacks of the prior art, the present inventors have long studied and have made a great deal of practical use to propose the technical solution of the present application. The application will be more fully understood from the following detailed description, which should be read in conjunction with the accompanying drawings. Detailed embodiments of the present application are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the application, which may be embodied in various forms. Therefore, specific functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present application in virtually any appropriately detailed embodiment.

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 contradicts or is inconsistent with a definition set forth in the other publications, the definition used herein controls.

As used herein, the terms "selected from", "comprised of …" and "comprising" are synonymous. As used herein, the terms "comprise," "include," "have," "contain," 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 list of elements is not necessarily limited to only those elements recited in the list, but may include other elements not expressly listed but inherent to such composition, process, method, article, or apparatus.

Where an equivalent, concentration, or other value or parameter gives a list of ranges, preferred ranges or 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 the like. If a numerical range is recited in the specification, unless otherwise indicated, that range is intended to include the endpoints thereof, and all integers and fractions within the range.

Furthermore, unless expressly stated otherwise, the term "or" means an inclusive "or" rather than an exclusive "or". For example, condition A "or" B is satisfied in any of the following cases: 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).

Likewise, the indefinite articles "a" and "an" preceding an element or component herein are intended to be interpreted as non-limiting with respect to the number of instances (i.e. the number of occurrences) of the element or component, unless the context clearly indicates otherwise. Accordingly, the terms "a," "an," and "the" are to be construed as including one or at least one, and the singular forms of the elements or components also include plural forms.

Several terms herein are defined as follows:

the term "polymerase chain reaction (polymerase chain reaction, PCR)" refers to a method for amplifying a specific nucleotide sequence (amplifier). PCR extends the primer on the template by virtue of the activity of the nucleic acid polymerase to obtain an amplifier. Preferably, the nucleic acid polymerase has thermostable properties.

The term "expression" refers to the process by which DNA is transcribed to form mRNA and/or the process by which transcribed mRNA is further translated into a peptide, polypeptide, or protein.

As one aspect of the technical scheme of the application, the functional DNA hydrogel is prepared by crosslinking a maleimide group-containing high molecular compound, a thiol group-containing high molecular compound, dopamine and thiol-modified DNA.

In some embodiments, the maleimide group-containing polymer compound or thiol group-containing polymer compound is multi-armed, and for example, may be preferably four-armed or eight-armed, and the like.

Further, the maleimide group-containing polymer compound or the thiol group-containing polymer compound is four-armed.

In some embodiments, the maleimide group-containing polymeric compound may include a four-arm polyethylene glycol maleimide, which may have an average number average molecular weight of 1000 to 20000g/mol.

In some embodiments, the thiol-group-containing polymer compound may include a four-arm polyethylene glycol thiol group, which may have an average molecular weight of 1000 to 20000g/mol.

In some embodiments, the thiol-modified DNA is amplified by PCR reactions using plasmid DNA containing the target protein gene and 5'utr and 3' utr sequences necessary for protein expression as templates, and using 5 'end-modified oligonucleotides as primers to synthesize a DNA product with thiol modification at the 5' end.

In some embodiments, the functionalized DNA hydrogel has a high mechanical strength, is not breakable, and has an elastic modulus (G') of 300 to 1200Pa in the frequency range of 0.1 to 5 Hz.

In conclusion, the functional DNA hydrogel provided by the application has high mechanical strength, is not easy to break, can protect a template, and further improves the protein expression quantity; and the hydrogel can be reused, so that the cost required by amplification products is reduced, and the protein expression cost is reduced.

In another aspect of embodiments of the present application, there is provided a method of preparing a functionalized DNA hydrogel comprising:

and (3) crosslinking a uniform mixed system containing the maleimide group-containing high molecular compound, the thiol group-containing high molecular compound, the dopamine and the thiol-modified DNA at room temperature to obtain the functionalized DNA hydrogel.

In some embodiments, the maleimide group-containing polymer compound or thiol group-containing polymer compound is multi-armed, and for example, may be preferably four-armed or eight-armed, and the like.

Further, the maleimide group-containing polymer compound or the thiol group-containing polymer compound is four-armed.

In some embodiments, the maleimide group-containing polymeric compound may include a four-arm polyethylene glycol maleimide, which may have an average number average molecular weight of 1000 to 20000g/mol.

Further, the concentration of the maleimide group-containing polymer compound in the homogeneously mixed system is 0.1 to 30wt%.

Further, the pH value of the maleimide group-containing polymer compound is 3.5 to 7.5.

In some embodiments, the thiol-group-containing polymer compound may include a four-arm polyethylene glycol thiol group, which may have an average molecular weight of 1000 to 20000g/mol.

Further, the concentration of the thiol-group-containing polymer compound in the homogeneous mixed system is 0.1 to 30wt%.

Further, the pH value of the thiol-group-containing polymer compound is 3.5 to 7.5.

Further, the concentration of thiol-modified DNA in the homogeneous mixed system is 5-50 ng/. Mu.L.

Further, the concentration of the dopamine in the uniform mixed system is 4-6wt%.

The preparation method of the functionalized DNA hydrogel provided by the application has the following mechanism: firstly, the inventor uses quadrifilar polyethylene glycol sulfhydryl, quadrifilar polyethylene glycol maleimide and dopamine (structural formulas are shown in formulas (1), (2) and (3)) as polymerization monomers, and combines DNA modified by functional groups to form the functional DNA hydrogel. The functionalized DNA hydrogel prepared by the method is simple to prepare, low in cost and high in feasibility. Meanwhile, the hydrogel can protect the template and improve the protein expression quantity; and the hydrogel can be reused, so that the cost required by amplification products is reduced.

In some preferred embodiments, the specific steps of the preparation method of the present application comprise:

plasmid DNA containing target protein gene and 5'UTR and 3' UTR sequences necessary for protein expression is used as template, oligonucleotide with 5 'end modified sulfhydryl is used as primer, DNA product with 5' end modified sulfhydryl is amplified through PCR reaction, the PCR amplified product is mixed with dopamine, and then mixed with quadrifilar polyethylene glycol sulfhydryl according to a certain proportion, and then further mixed with quadrifilar polyethylene glycol maleimide for reaction, thus forming porous hydrogel, and the porous hydrogel is applied to cell-free protein synthesis. The four-arm polyethylene glycol maleimide and the four-arm polyethylene glycol mercapto are used as biological high polymer materials, have hydrophilicity, good biocompatibility and low cytotoxicity, and can not inhibit the protein synthesis of a CFPS system; dopamine, a neurotransmitter, is also not inhibitory and cytotoxic. The hydrogel material is applied to cell-free protein production, so that the thiol-modified PCR product can be protected, and the local effective concentration of the template can be increased. The application realizes the improvement of the protein yield, the template can be reused for a plurality of times, and the reaction cost is reduced.

In some specific embodiments, the crosslinking reaction includes an addition reaction and a polymerization reaction. Wherein, C-C double bond in the four-arm polyethylene glycol maleimide can generate addition reaction with sulfhydryl; the amino in the dopamine can be subjected to N-acylation reaction with C-O double bonds in the four-arm polyethylene glycol maleimide and the four-arm polyethylene glycol mercapto; moreover, a reaction between mercapto groups may occur to form disulfide bonds.

Further, the time of the crosslinking reaction is 1s-12h, and the glue forming speed is high during preparation and is generally within 60 seconds.

Further, the preparation of the functionalized DNA hydrogel can be performed at room temperature.

Further, the functionalized DNA hydrogel is prepared without conditions such as initiation, coagulation and the like.

In some more specific embodiments, the method of preparing the functionalized DNA hydrogel comprises the steps of:

the four-arm polyethylene glycol sulfhydryl (average molecular weight: 1000-20000 g/mol, concentration: 0.1% -30%), four-arm polyethylene glycol maleimide (average molecular weight: 1000-20000 g/mol, concentration: 0.1% -30%), dopamine (molecular weight: 153g/mol, concentration 5%), DNA modified with functional group (concentration: 5-50 ng/. Mu.L) and water are mixed uniformly, and can be reacted within 1s-12h to form the functional DNA hydrogel.

In conclusion, the preparation process of the functionalized DNA hydrogel provided by the application is simple, quick and low in cost, does not need conditions such as initiation, coagulation acceleration and the like, has high gel forming speed in preparation, generally within 60 seconds, has high feasibility and can be used for cell-free protein expression.

In another aspect of embodiments of the present application, there is provided a functionalized DNA hydrogel prepared by the foregoing method.

Another aspect of embodiments of the present application also provides the use of the functionalized DNA hydrogels described above in the field of cell-free protein expression.

Furthermore, the functionalized DNA hydrogel can be used for cell-free protein expression, and can protect a linear template and further improve the protein yield.

Accordingly, another aspect of an embodiment of the present application also provides a method for synthesizing a protein, comprising:

providing the functionalized DNA hydrogel described previously;

the functionalized DNA hydrogel is used for synthesizing protein.

In some embodiments, the method specifically comprises: the cell extract, the energy buffer solution and the hydrogel are mixed according to the volume ratio of (10-100): (10-100): (1-50), and shaking the obtained mixed solution for 0.5-48 hours at the temperature of 0-37 ℃ and the rotating speed of 0-2000rpm to obtain a solution containing expressed protein, and further synthesizing the protein.

In some embodiments, the cell extract is derived from a prokaryotic cell or a eukaryotic cell.

In some embodiments, the energy buffer comprises three components, an amino acid mixture, a reaction buffer, and an energy replenishment solution, respectively.

Further, the amino acid mixed solution is a mixed solution with equimolar concentration, which is prepared by adding deionized water into leucine, isoleucine, methionine, tyrosine, glycine, alanine, amino acid, serine, threonine, half-sarcosine, asparaffinine, phenylalanine, tryptophan, aspartic acid, glutamic acid, lysine, arginine and histidine, and the molar concentration is 1-4 mmol/L.

Further, the reaction buffer solution consists of 1-3% of polyethylene glycol 8000, 1-3 mmol/L dithiothreitol, 60-80 mmol/L potassium glutamate, 3-6 mmol/L magnesium glutamate and 14-18 mmol/L sucrose, and the solvent is deionized water.

Further, the energy replenishing solution comprises 0.8-1 mmol/L guanine-5 ' -triphosphate, 1-2 mmol/L adenine nucleoside triphosphate, 0.8-1 mmol/L cytidine triphosphate, 0.8-1 mmol/L uridine triphosphate, 50-60 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 0.4-1 mmol/L spermidine, 0.3-0.4 mmol/L nicotinamide adenine dinucleotide, 0.6-0.9 mmol/L adenosine-3 ',5' -cyclic monophosphate, 0.1-0.2 mg/ml transport ribonucleic acid, 0.03-0.07 mmol/L folinic acid, 0.2-0.3 mmol/L coenzyme A and 10-40 mmol/L3-phosphoglycerate, and deionized water is used as a solvent.

In some more specific embodiments, the method of synthesizing a protein comprises the steps of:

the cell extract, the energy buffer solution and the hydrogel are mixed according to the volume ratio of (10-100): (10-100): (1-50), the total volume is 10-1000 mu L, the rest part is filled with water, the mixed system is always placed in a constant temperature reaction vessel, and the mixed system is oscillated for 0.5-48 hours at the temperature of 0-37 ℃ and the rotating speed of 0-2000rpm, so as to obtain the solution containing the expressed protein.

By means of the technical scheme, the functional DNA hydrogel material is applied to cell-free protein production, can protect a sulfhydryl modified PCR product and can increase the local effective concentration of a template. The application realizes the improvement of the protein yield, the template can be reused for a plurality of times, and the reaction cost is reduced.

The technical solution of the present application will be described in further detail below with reference to a number of preferred embodiments and accompanying drawings, and it is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application. The implementation conditions used in the following examples may be further adjusted according to actual needs, and the implementation conditions not specified are generally those in routine experiments.

EXAMPLE 1 preparation of functionalized DNA hydrogels and use for cell-free protein expression

1. Preparing a template: the pID-1-GFP plasmid is used as an amplification template, a single-stranded oligonucleotide fragment with sulfhydryl modification at the 5' end is used as a primer, PCR reaction is carried out by using the template, the primer, DNA polymerase and other PCR requisite conditions to obtain a PCR product of the target gene GFP, and the PCR product contains a gene expressing GFP fluorescent protein and a promoter, RBS, 5' -UTR and 3' -UTR region necessary for gene transcription and translation and is used as a template for protein synthesis.

2. Preparation of functionalized DNA hydrogels: mu.l of dopamine (final concentration: 5%, pH=7), thiol-modified PCR product, water, 0.4. Mu.l of four-arm polyethylene glycol thiol (final concentration: 2%, average molecular weight: 20000, pH=4), 0.4. Mu.l of four-arm polyethylene glycol maleimide (final concentration: 2%, average molecular weight: 20000, pH=4) and a total of 10. Mu.l of the system were added to a PCR tube, respectively, in this order, each of the components was thoroughly mixed uniformly after the addition, and the reaction was allowed to proceed quickly at room temperature to give a porous DNA hydrogel, and the obtained functionalized DNA hydrogel was used for the subsequent cell-free protein expression.

3. Cell-free protein expression of functionalized DNA hydrogels

The cell-free expression system components are as follows: the preparation method comprises the steps of adding 100 ng/. Mu.l of functionalized DNA hydrogel containing GFP fluorescent protein genes, 10mM phosphate buffer, 1.2mM ATP, 0.85mM UTP, 0.85mM CTP, 0.85mM GTP, 280mM potassium glutamate, 8mM magnesium glutamate, 2.7mM potassium oxalate, 2mM amino acid mixture and 25% of escherichia coli extract by volume into a PCR tube, uniformly mixing, vibrating at 30 ℃ and 1000rpm by using a constant temperature mixer, taking out after about 12 hours, transferring an expression product into a black hole plate, measuring fluorescence intensity by using an enzyme-labeled instrument under the conditions of 485nm of exciting light, 535nm of emitting light and 0.1s of exposure time, and reflecting the expression quantity of green fluorescent protein. The results show (figure 2), under the same condition, compared with the common linear template (PCR product), the protein expression quantity of the functionalized DNA hydrogel containing 0.8% and 2% of the four-arm polyethylene glycol provided by the patent is obviously better than that of the PCR product serving as the template.

Test example 1: reuse of functionalized DNA hydrogels

In the CFPS reaction system, hydrogel containing template DNA, lysate and energy buffer solution are added to complete fluorescence expression, and then fresh lysate is supplemented to reuse hydrogel, so that repeated protein production can be realized, as shown in FIG. 3, which shows the repeated use condition of the functionalized DNA hydrogel, and the prepared DNA hydrogel can be reused for tens of times.

Example 2: effects of different concentrations, molecular weights of polymers and reaction times on cell-free protein expression

1. Results of DNA hydrogel protein expression formed by four-arm polyethylene glycol sulfhydryl and four-arm polyethylene glycol maleimide with different concentrations and DNA template

The four-arm polyethylene glycol sulfhydryl groups (the final concentration is 0.1%, 0.5%, 0.8%, 1%, 2%, 5%, 25%, 30%) and the four-arm polyethylene glycol maleimide (the final concentration is 0.1%, 0.5%, 0.8%, 1%, 2%, 5%, 25%, 30%), dopamine (the final concentration is 4-6%), the sulfhydryl modified PCR product and water are mixed uniformly to obtain porous DNA hydrogel at room temperature, the expression level of the hydrogel proteins at different concentrations is shown in figure 4, the concentration of the formed functional DNA hydrogel template is the same, the dopamine concentration is 5%, and the number average molecular weight of the four-arm polyethylene glycol sulfhydryl groups and the four-arm polyethylene glycol maleimide is 20000. Wherein a represents the fluorescence intensity of the expression of a functional DNA hydrogel protein formed by 0.5% of quadrifilar polyethylene glycol mercapto group, 0.5% of quadrifilar polyethylene glycol maleimide and the like; b represents the fluorescence intensity of the expression of the functional DNA hydrogel protein formed by 0.8% of quadrifilar polyethylene glycol mercapto group, 0.8% of quadrifilar polyethylene glycol maleimide and the like; c represents the fluorescence intensity of the expression of the functional DNA hydrogel protein formed by 1% of four-arm polyethylene glycol mercapto group, 1% of four-arm polyethylene glycol maleimide and the like; d represents the fluorescence intensity of the functional DNA hydrogel protein expression formed by 2% of four-arm polyethylene glycol mercapto groups, 2% of four-arm polyethylene glycol maleimide and the like; e represents the fluorescence intensity of the functional DNA hydrogel protein expression formed by 5% of quadrifilar polyethylene glycol mercapto groups, 5% of quadrifilar polyethylene glycol maleimide and the like; f represents the fluorescence intensity of the functional DNA hydrogel protein expression formed by 25% of quadrifilar polyethylene glycol mercapto groups, 25% of quadrifilar polyethylene glycol maleimide and the like; g represents the fluorescence intensity of the template protein expression in the solution state.

2. Results of expression of DNA hydrogel proteins formed by four-arm polyethylene glycol sulfhydryl groups and four-arm polyethylene glycol maleimide with different molecular weights and DNA templates

Four-arm polyethylene glycol sulfhydryl groups (with final concentrations of 2%, 5% and 25% respectively and number average molecular weights of 1000%, 5000 and 20000) and four-arm polyethylene glycol maleimide (with final concentrations of 2%, 5% and 25% respectively), dopamine, sulfhydryl modified PCR products and water, wherein the total system is 10 mu L, the porous DNA hydrogel is obtained at room temperature, the expression level of hydrogel proteins formed by polymers with different molecular weights is shown in figure 5, the concentration of the formed functionalized DNA hydrogel templates is the same, and the concentration of dopamine is 5%. Wherein A represents the fluorescence intensity of the expression of the functionalized DNA hydrogel protein formed by four-arm polyethylene glycol mercapto group, four-arm polyethylene glycol maleimide and the like, wherein the number average molecular weight is 5000 and the concentration is 2 percent; b represents the fluorescence intensity of the expression of the functionalized DNA hydrogel protein formed by four-arm polyethylene glycol mercapto groups, four-arm polyethylene glycol maleimide and the like, wherein the number average molecular weight of the functionalized DNA hydrogel protein is 20000, and the concentration of the functionalized DNA hydrogel protein is 2 percent; c represents the fluorescence intensity of the expression of the functionalized DNA hydrogel protein formed by four-arm polyethylene glycol mercapto groups, four-arm polyethylene glycol maleimide and the like, wherein the number average molecular weight is 5000 and the concentration is 5 percent; d represents the fluorescence intensity of the expression of the functionalized DNA hydrogel protein formed by four-arm polyethylene glycol mercapto groups, four-arm polyethylene glycol maleimide and the like, wherein the number average molecular weight of the functionalized DNA hydrogel protein is 20000, and the concentration of the functionalized DNA hydrogel protein is 5%; e represents the fluorescence intensity of the expression of the functionalized DNA hydrogel protein formed by four-arm polyethylene glycol mercapto groups, four-arm polyethylene glycol maleimide and the like, wherein the number average molecular weight is 5000 and the concentration is 25 percent; f represents the fluorescence intensity of the expression of the functionalized DNA hydrogel protein formed by four-arm polyethylene glycol mercapto groups, four-arm polyethylene glycol maleimide and the like, wherein the number average molecular weight of the protein is 20000, and the concentration of the protein is 25%; g represents the fluorescence intensity of the template protein expression in the solution state.

3. Influence of different pH values on gel formation time of reaction of four-arm polyethylene glycol mercapto group and four-arm polyethylene glycol maleimide

Four-arm polyethylene glycol sulfhydryl groups (the final concentration is 2%, the pH values are 7.5, 7, 6, 5, 4 and 3.5 respectively), four-arm polyethylene glycol maleimide (the final concentration is 2%, the pH values are 7.5, 7, 6, 5, 4 and 3.5 respectively), dopamine, and sulfhydryl modified PCR products and water are mixed uniformly for 1s-12h at room temperature to obtain the porous DNA hydrogel through reaction, wherein the total volume of the system is 10 mu L.

Test example 2 determination of mechanical Properties of functionalized DNA hydrogels

Dopamine (final concentration 5%, molecular weight 153, ph=7), thiol-modified PCR products, water, four-arm polyethylene glycol thiol (final concentration 2%, 5%, number average molecular weight 20000, ph=4) and four-arm polyethylene glycol maleimide (final concentration 2%, 5%, number average molecular weight 20000, ph=7) were added to a total of 50 μl of PCR tubes, each component was thoroughly mixed after addition, and the reaction was allowed to give a porous DNA hydrogel. The relationship between the elastic modulus (G ') and the viscous modulus (G') of DNA hydrogels was measured using a rotameter (see FIG. 7), and when the elastic modulus was greater than the viscous modulus, it was demonstrated that the polymer was a solid, and vice versa, a liquid. As shown in FIG. 7, the DNA hydrogel has G 'which is always larger than G' in the tested frequency range, and shows good elastic mechanical properties of the hydrogel.

In summary, by the technical scheme, the preparation process of the functionalized DNA hydrogel provided by the application is simple, rapid and low in cost, does not need conditions such as initiation, coagulation acceleration and the like, has high gelling speed in preparation, generally within 60 seconds, has high feasibility and can be used for cell-free protein expression; meanwhile, the functional DNA hydrogel disclosed by the application has higher mechanical strength and is not easy to break, so that not only can the PCR product modified by sulfhydryl groups be protected, but also the local effective concentration of a template can be increased, and the protein expression quantity is further improved; the hydrogel can be reused, so that the protein expression cost is reduced.

The various aspects, embodiments, features and examples of the application are to be considered in all respects as illustrative and not intended to limit the application, the scope of which is defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed application.

The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the present application.

Throughout this disclosure, where a composition is described as having, comprising, or including a particular component, or where a process is described as having, comprising, or including a particular process step, it is contemplated that the composition of the teachings of the present application also consist essentially of, or consist of, the recited component, and that the process of the teachings of the present application also consist essentially of, or consist of, the recited process step.

Unless specifically stated otherwise, the use of the terms "comprising (include, includes, including)", "having (has, has or has)" should generally be understood to be open-ended and not limiting.

The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. Furthermore, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise. In addition, where the term "about" is used prior to a magnitude, the present teachings include the particular magnitude itself, unless specifically stated otherwise.

It should be understood that the order of steps or order in which a particular action is performed is not critical, as long as the present teachings remain operable. Furthermore, two or more steps or actions may be performed simultaneously.

In addition, the inventors have conducted experiments with other materials, process operations, and process conditions as described in this specification with reference to the foregoing examples, and have all obtained desirable results.

While the application has been described with reference to an illustrative embodiment, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the application. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the application without departing from the scope thereof. Therefore, it is intended that the application not be limited to the particular embodiment disclosed for carrying out this application, but that the application will include all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated any use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.

Claims (19)

1. The functional DNA hydrogel is characterized by being prepared from a maleimide group-containing high molecular compound, a thiol group-containing high molecular compound, dopamine and thiol-modified DNA through a crosslinking reaction;

the crosslinking reaction is carried out at room temperature;

the crosslinking reaction includes an addition reaction and a polymerization reaction;

the addition reaction of the C-C double bond and the sulfhydryl in the maleimide group-containing high molecular compound;

taking the maleimide group-containing high molecular compound, the thiol group-containing high molecular compound and dopamine as polymerization monomers, and carrying out N-acylation reaction on amino groups in the dopamine, the maleimide group-containing high molecular compound and C-O double bonds in the thiol group-containing high molecular compound; the sulfhydryl groups react to form disulfide bonds;

wherein the macromolecular compound containing maleimide group comprises four-arm polyethylene glycol maleimide;

the thiol-containing high molecular compound comprises a four-arm polyethylene glycol thiol;

the average number average molecular weight of the four-arm polyethylene glycol maleimide is 1000-20000 g/mol;

the average molecular weight of the four-arm polyethylene glycol mercapto is 1000-20000 g/mol.

2. The functionalized DNA hydrogel of claim 1, wherein: and the elastic modulus of the functionalized DNA hydrogel is 300-1200 Pa in the frequency range of 0.1-5 Hz.

3. A method of preparing a functionalized DNA hydrogel according to claim 1 or 2, comprising:

and (3) crosslinking a uniform mixed system containing the maleimide group-containing high molecular compound, the thiol group-containing high molecular compound, the dopamine and the thiol-modified DNA at room temperature to obtain the functionalized DNA hydrogel.

4. A method of preparation according to claim 3, characterized in that: the crosslinking reaction includes an addition reaction and a polymerization reaction; and/or the time of the crosslinking reaction is 1s-12 h.

5. A method of preparation according to claim 3, characterized in that: the time of the crosslinking reaction is less than 60 s.

6. A method of preparation according to claim 3, characterized in that: the pH value of the maleimide group-containing high molecular compound is 3.5-7.5.

7. A method of preparation according to claim 3, characterized in that: the concentration of the thiol-containing polymer compound in the uniform mixed system is 0.1-30wt%.

8. A method of preparation according to claim 3, characterized in that: the pH value of the thiol-containing polymer compound is 3.5-7.5.

9. A method of preparation according to claim 3, characterized in that: the concentration of the sulfhydryl modified DNA in the uniform mixed system is 5-50 ng/MuL.

10. A method of preparation according to claim 3, characterized in that: the concentration of the dopamine in the uniform mixed system is 4-6wt%.

11. Use of the functionalized DNA hydrogel of claim 1 or 2 in the field of cell-free protein expression for non-therapeutic purposes.

12. A method of synthesizing a protein, characterized by: comprising

Providing a functionalized DNA hydrogel according to claim 1 or 2;

the functionalized DNA hydrogel is used for synthesizing protein.

13. The method according to claim 12, wherein: specifically comprises the steps of mixing a cell extract, an energy buffer solution and hydrogel according to the volume ratio of (10-100): (10-100): (1-50), and vibrating the obtained mixed solution for 0.5-48h at the temperature of 0-37 ℃ and the rotating speed of 0-2000rpm to obtain a solution containing expressed protein, thereby synthesizing the protein.

14. The method according to claim 13, wherein: the cell extract is derived from a prokaryotic cell or a eukaryotic cell.

15. The method according to claim 13, wherein: the energy buffer solution comprises an amino acid mixed solution, a reaction buffer solution and an energy supplementing solution.

16. The method according to claim 15, wherein: the amino acid mixture comprises leucine, isoleucine, methionine, tyrosine, glycine, alanine, arginine, serine, threonine, half-sarcosine, asparagusamine, phenylalanine, tryptophan, aspartic acid, glutamic acid, lysine, arginine and histidine.

17. The method according to claim 15, wherein: the molar concentration of the amino acid mixed solution is 1-4 mmol/L.

18. The method according to claim 15, wherein: the reaction buffer solution comprises 1-3% of polyethylene glycol 8000, 1-3 mmol/L dithiothreitol, 60-80 mmol/L potassium glutamate, 3-6 mmol/L magnesium glutamate and 14-18 mmol/L sucrose, and the solvent is deionized water.

19. The method according to claim 15, wherein: the energy supplementing liquid comprises 0.8-1 mmol/L guanine-5 ' -triphosphate, 1-2 mmol/L adenine nucleoside triphosphate, 0.8-1 mmol/L cytidine triphosphate, 0.8-1 mmol/L uridine triphosphate, 50-60 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 0.4-1 mmol/L spermidine, 0.3-0.4 mmol/L nicotinamide adenine dinucleoside, 0.6-0.9 mmol/L adenosine-3 ',5' -cyclized monophosphate, 0.1-0.2 mg/mL transport ribonucleic acid, 0.03-0.07 mmol/L folinic acid, 0.2-0.3 mmol/L coenzyme A and 10-40 mmol/L3-phosphoglycerate, and the solvent is deionized water.