CN112661987A - Functionalized DNA hydrogel, and preparation method and application thereof - Google Patents
Functionalized DNA hydrogel, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a functionalized DNA hydrogel, a preparation method and application thereof. The preparation method comprises the following steps: and carrying out crosslinking reaction on a uniformly mixed system containing a maleimide-containing macromolecular compound, a sulfhydryl-containing macromolecular compound, dopamine and sulfhydryl-modified DNA at room temperature to obtain the functionalized DNA hydrogel. The preparation process of the functional DNA hydrogel provided by the invention is simple and rapid, the cost is low, conditions such as initiation and procoagulant are not needed, the gelling speed is rapid during preparation, generally within 60 seconds, the practicability is high, and the functional DNA hydrogel can be used for cell-free protein expression; meanwhile, the functional DNA hydrogel has high mechanical strength and is not easy to break, so that a sulfhydryl-modified PCR product can be protected, the local effective concentration of a template can be increased, and the protein expression amount is further improved; and the hydrogel can be repeatedly used, so that the protein expression cost is reduced.
Description
Technical Field
The invention relates to a novel hydrogel, in particular to a functional DNA hydrogel for cell-free protein synthesis (CFPS) and a preparation method and application thereof, belonging to the technical field of hydrogels.
Background
At present, protein drugs such as antibodies, polypeptides and the like are more and more widely used in disease treatment, but the problem faced at present is that antibody drugs such as targeting drugs for treating cancer are expensive, and many patients often abandon treatment because the price cannot be borne. The cost of antibody drugs is mainly due to their high cost. At present, the main mode of protein medicine production is that living cells are produced and then extracted, the operation steps are multiple, cell culture is needed, and pollution is easily caused in the culture process, so that the production cost of protein medicines is increased. The advent of the cell-free protein synthesis (CFPS) system has provided a possible solution to the above-mentioned problem. The cell-free protein synthesis is an in vitro life simulation system independent of a complete cell structure, realizes the in vitro expression of target protein by utilizing a protein factor, enzyme and protein synthesis machine in a cell extract and taking exogenous DNA or mRNA as a template to supplement amino acid, energy and the like, provides a powerful platform for the in vitro synthesis of protein and peptide, and provides a non-biological way for efficiently expressing protein. CFPS breaks through the physiological limitation of cells, and has the unique advantage 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 a CFPS system is directly utilized for protein expression, so that the protein synthesis time is shortened. However, since the linear template is susceptible to degradation by nucleases in cell-free expression systems, the template concentration is reduced, thereby reducing the protein expression efficiency.
At present, researchers in the industry mainly protect the linear template from two directions to improve the protein expression efficiency in CFPS. One direction is to inhibit or reduce nuclease activity; the second direction is that protecting linear DNA reduces exposure to nucleases reduces template degradation increasing local concentration of template.
Ahn et al used a knock-out strain encoding endonuclease E gene to prepare cell-free extracts, delaying the degradation of mRNA molecules, and the protein level expressed from the PCR-amplified template became comparable to conventional plasmid-based reactions. However, there is a problem in that the strain grows slowly and the cycle is long.
Gams protein from bacteriophage lambda, whose truncated form of GamS has been used for cell-free protein expression to inhibit nuclease activity and to achieve protein expression levels approaching plasmid levels, has been identified as a potent inhibitor of RecBCD nuclease, but has a problem in that it requires synthesis and purification of this protein in bacteria, which is cumbersome to handle.
The Luo topic group reports a DNA clay hybrid hydrogel that mimics the cell sealing function through electrostatic interaction between DNA and clay, and the clay hydrogel can provide an effective sealing environment and protect genes from nuclease. When they further used this mixed hydrogel in a CFPS system, the yield of protein was 6 times that of a liquid phase system. However, the mechanical strength of the clay hydrogel is low, and the clay hydrogel is difficult to recover after expression, which makes recycling difficult.
In conclusion, the direct genetic modification of the strain in the prior art leads to slow growth and longer period of the strain; the method for synthesizing the RecBCD nuclease inhibitor needs the synthesis and purification of protein, and the operation is complicated; the clay hydrogel has low mechanical strength and is difficult to recycle.
Disclosure of Invention
The invention mainly aims to provide a functionalized DNA hydrogel and a preparation method thereof, so as to overcome the defects in the prior art.
Another object of the present invention is to provide the use of the functionalized DNA hydrogel for the expression of cellular proteins.
In order to achieve the purpose, the invention adopts the following technical scheme:
the embodiment of the invention provides a functionalized DNA hydrogel which is prepared by cross-linking reaction of a macromolecular compound containing maleimide groups, a macromolecular compound containing sulfydryl groups, dopamine and DNA modified by sulfydryl groups.
In some embodiments, the maleimide group-containing polymeric compound or thiol group-containing polymeric compound is multi-armed.
Further, the polymer compound containing maleimide groups comprises four-arm polyethylene glycol maleimide.
Further, the thiol-group-containing polymer compound includes a four-arm polyethylene glycol thiol group.
The embodiment of the invention also provides a preparation method of the functionalized DNA hydrogel, which comprises the following steps:
and carrying out crosslinking reaction on a uniformly mixed system containing a maleimide-containing macromolecular compound, a thiol-containing macromolecular compound, dopamine and thiol-modified DNA at room temperature to obtain the functionalized DNA hydrogel.
The embodiment of the invention also provides the functionalized DNA hydrogel prepared by the method.
The embodiment of the invention also provides application of the functionalized DNA hydrogel in the field of cell-free protein synthesis (CFPS).
Accordingly, embodiments of the present invention also provide a method for synthesizing a protein, which includes:
providing the aforementioned functionalized DNA hydrogel;
and synthesizing protein by using the functionalized DNA hydrogel.
In some embodiments, the method specifically comprises: mixing a cell extract, an energy buffer solution and hydrogel according to a volume ratio of (10-100): (10-100): (1-50), and shaking the obtained mixed solution for 0.5-48h at 0-37 ℃ and 0-2000rpm to obtain a solution containing the expressed protein, thereby synthesizing the protein.
Compared with the prior art, the invention has the beneficial effects that:
1) the preparation process of the functionalized DNA hydrogel provided by the invention is simple and rapid, has low cost, does not need conditions such as initiation and procoagulant, has high gelling speed in preparation, is generally within 60 seconds, has high feasibility of implementation, and can be used for cell-free protein expression;
2) the functional DNA hydrogel provided by the invention has higher mechanical strength, is not easy to break, can protect the template, and further improves the protein expression amount; the hydrogel can be repeatedly used, so that the cost required by an amplification product is reduced, and the protein expression cost is reduced;
3) the functionalized DNA hydrogel material provided by the invention 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 invention realizes the improvement of protein yield, the template can be repeatedly used 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 needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIGS. 1a and 1b are schematic diagrams of a hydrogel reaction between a polymer and a modifying group on DNA in an exemplary embodiment of the invention;
FIG. 2 is a schematic diagram showing the results of the difference between the protein expression levels of the functionalized DNA hydrogel and the liquid phase system in example 1 of the present invention;
FIG. 3 is a graph showing the results of repeated use of the functionalized DNA hydrogel in test example 1 of the present invention;
FIG. 4 is a graph showing the expression levels of the functionalized DNA hydrogel proteins at different concentrations in example 2 of the present invention;
FIG. 5 is a graph showing the expression levels of the functionalized DNA hydrogel proteins formed from polymers of different molecular weights in example 2 of the present invention;
FIG. 6 is a graph showing the effect of different pH values on gel formation time of the reaction of a four-arm PEG thiol and a four-arm PEG maleimide in example 2 of the present invention;
FIG. 7 is a graph showing the relationship between the elastic modulus (G ') and the viscous modulus (G') of the functionalized DNA hydrogel in test example 2 of the present invention.
Detailed Description
As described above, in view of the defects of the prior art, the inventors of the present invention have made extensive studies and extensive practices to propose a technical solution of the present invention. The present invention 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 invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can 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 invention 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 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.
In one aspect of the present invention, a functionalized DNA hydrogel is prepared by crosslinking a maleimide-containing polymer compound, a thiol-containing polymer 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, four-armed or eight-armed, etc. may be preferable.
Further, the maleimide group-containing polymer compound or the thiol group-containing polymer compound is four-armed.
In some embodiments, the maleimide group-containing polymer compound may include a four-arm polyethylene glycol maleimide, and the average number average molecular weight thereof may be 1000 to 20000 g/mol.
In some embodiments, the thiol-group-containing polymer compound may include a four-arm thiol group of polyethylene glycol, and the average molecular weight thereof may be 1000 to 20000 g/mol.
In some embodiments, the thiol-modified DNA is synthesized by PCR amplification using a plasmid DNA containing the target protein gene and 5' UTR and 3' UTR sequences necessary for protein expression as a template and 5' thiol-modified oligonucleotide as a primer.
In some embodiments, the functionalized DNA hydrogel has high mechanical strength and is not easy to break, and the elastic modulus (G') of the functionalized DNA hydrogel is 300-1200 Pa within the frequency range of 0.1-5 Hz.
In conclusion, the functional DNA hydrogel provided by the invention has high mechanical strength and is not easy to break, and can protect the template and further improve the protein expression amount; 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 the embodiments of the present invention, there is provided a method for preparing a functionalized DNA hydrogel, including:
and carrying out crosslinking reaction on a uniformly mixed system containing a maleimide-containing macromolecular compound, a thiol-containing macromolecular compound, dopamine and 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, four-armed or eight-armed, etc. may be preferable.
Further, the maleimide group-containing polymer compound or the thiol group-containing polymer compound is four-armed.
In some embodiments, the maleimide group-containing polymer compound may include a four-arm polyethylene glycol maleimide, and the average number average molecular weight thereof may be 1000 to 20000 g/mol.
Further, the concentration of the polymer compound containing maleimide groups in the uniform mixing system is 0.1-30 wt%.
Further, the pH value of the polymer compound containing maleimide groups is 3.5-7.5.
In some embodiments, the thiol-group-containing polymer compound may include a four-arm thiol group of polyethylene glycol, and the average molecular weight thereof may be 1000 to 20000 g/mol.
Further, the concentration of the thiol-containing polymer compound in the uniform mixing system is 0.1-30 wt%.
Further, the pH value of the thiol-group-containing polymer compound is 3.5-7.5.
Further, the concentration of the sulfhydryl-modified DNA in the uniform mixing system is 5-50 ng/muL.
Further, the concentration of the dopamine in the uniformly mixed system is 4-6 wt%.
The mechanism of the preparation method of the functionalized DNA hydrogel provided by the application is as follows: firstly, the applicant utilizes four-arm polyethylene glycol sulfydryl, four-arm polyethylene glycol maleimide and dopamine (structural formulas are respectively shown in the following formulas (1), (2) and (3)) as polymerization monomers, and combines DNA modified by functional groups to form the functionalized DNA hydrogel. The functional DNA hydrogel prepared by the method is simple to prepare, low in price and high in practicability. Meanwhile, the hydrogel can protect the template and improve the protein expression; and the hydrogel can be reused, so that the cost required by amplification products is reduced.
In some preferred embodiments, the preparation method of the present invention comprises the following specific steps:
the method comprises the steps of taking plasmid DNA containing target protein genes and 5 'UTR and 3' UTR sequences necessary for protein expression as a template, using 5 'end modified sulfhydryl oligonucleotide as a primer, carrying out PCR reaction amplification to synthesize a DNA product with a sulfhydryl modified 5' end, mixing the PCR amplification product with dopamine, mixing the mixture with a four-arm polyethylene glycol sulfhydryl according to a certain proportion, further mixing the mixture with a four-arm polyethylene glycol maleimide to react to form porous hydrogel, and applying the porous hydrogel to cell-free protein synthesis. The four-arm polyethylene glycol maleimide and the four-arm polyethylene glycol sulfydryl are used as biological polymer materials, have hydrophilicity, good biocompatibility and low cytotoxicity, and do not inhibit the protein synthesis of a CFPS system; dopamine, as a neurotransmitter, is also not inhibitory and cytotoxic. The hydrogel material is applied to cell-free protein production, so that the sulfhydryl-modified PCR product can be protected, and the local effective concentration of the template can be increased. The invention realizes the improvement of protein yield, the template can be repeatedly used 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, the C-C double bond in the four-arm polyethylene glycol maleimide can generate addition reaction with sulfydryl; the amino group in the dopamine can perform N-acylation reaction with the C-O double bond in the four-arm polyethylene glycol maleimide and the four-arm polyethylene glycol sulfydryl group; furthermore, the thiol groups can react with each other to form disulfide bonds.
Furthermore, the time of the crosslinking reaction is 1s-12h, and the gelling speed is high during preparation, generally within 60 seconds.
Further, the preparation of the functionalized DNA hydrogel can be performed under room temperature conditions.
Further, the functionalized DNA hydrogel does not need conditions such as initiation, procoagulant and the like when being prepared.
In some more specific embodiments, the method for preparing the functionalized DNA hydrogel comprises the following steps:
the four-arm polyethylene glycol sulfydryl (average molecular weight: 1000-20000 g/mol, concentration: 0.1-30%) and four-arm polyethylene glycol maleimide (average molecular weight: 1000-20000 g/mol, concentration: 0.1-30%), dopamine (molecular weight: 153g/mol, concentration: 5%), DNA (concentration: 5-50 ng/muL) for modifying the functional group and water are mixed uniformly, and the reaction can be finished within 1s-12h to form the functional DNA hydrogel.
In conclusion, the preparation process of the functionalized DNA hydrogel provided by the invention is simple and rapid, the cost is low, conditions such as initiation and procoagulant are not needed, the gelling speed is rapid in preparation, the gel formation is generally within 60 seconds, the feasibility of implementation is high, and the functionalized DNA hydrogel can be used for cell-free protein expression.
In another aspect of the embodiments of the present invention, there is also provided a functionalized DNA hydrogel prepared by the foregoing method.
In another aspect of the embodiments of the present invention, there is also provided a use of the aforementioned functionalized DNA hydrogel in the field of cell-free protein expression.
Further, the functionalized DNA hydrogel can be used for cell-free protein expression, can protect a linear template, and further improves the protein yield.
Accordingly, another aspect of the embodiments of the present invention also provides a method for synthesizing a protein, including:
providing the aforementioned functionalized DNA hydrogel;
and synthesizing protein by using the functionalized DNA hydrogel.
In some embodiments, the method specifically comprises: mixing a cell extract, an energy buffer solution and hydrogel according to a volume ratio of (10-100): (10-100): (1-50), and shaking the obtained mixed solution for 0.5-48h at 0-37 ℃ and 0-2000rpm to obtain a solution containing the expressed protein, thereby synthesizing the protein.
In some embodiments, the cellular extract is derived from a prokaryotic cell or a eukaryotic cell.
In some embodiments, the energy buffer comprises three components, which are an amino acid mixture, a reaction buffer, and an energy replenisher.
Furthermore, the amino acid mixed solution is a mixed solution with an equimolar concentration, which is prepared by adding leucine, isoleucine, methionine, tyrosine, glycine, alanine, valine, serine, threonine, hemisarcosine, asparagines, phenylalanine, tryptophan, aspartic acid, glutamic acid, lysine, arginine and histidine into deionized water, and the molar concentration is 1-4 mmol/L.
Further, the reaction buffer solution is composed 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 supplementing liquid comprises 0.8-1 mmol/L guanine-5 ' -triphosphate, 1-2 mmol/L adenosine 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-phosphoglyceride, and deionized water is used as a solvent.
In some more specific embodiments, the method of synthesizing a protein comprises the steps of:
mixing the cell extract, the energy buffer solution and the hydrogel according to the volume ratio of (10-100): (10-100): (1-50), the total volume is 10-1000 mu L, the insufficient part is filled with water, the mixed system is placed in a constant temperature reaction vessel, the temperature is 0-37 ℃, the rotation speed is 0-2000rpm, and the shaking is carried out for 0.5-48h, so as to obtain the solution containing the expression protein.
By the technical scheme, the functionalized 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 invention realizes the improvement of protein yield, the template can be repeatedly used for a plurality of times, and the reaction cost is reduced.
The technical solutions of the present invention will be described in further detail below with reference to several preferred embodiments and accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The conditions used in the following examples may be further adjusted as necessary, and the conditions used in the conventional experiments are not generally indicated.
Example 1 preparation of functionalized DNA hydrogels and use for cell-free protein expression
1. Preparing a template: pID-1-GFP plasmid is used as an amplification template, a single-stranded oligonucleotide fragment with a sulfhydryl modification at the 5' end is used as a primer, the template, the primer, DNA polymerase and other PCR essential conditions are utilized to carry out PCR reaction, and a PCR product of the target gene GFP is obtained, wherein the PCR product contains a gene for expressing GFP fluorescent protein, a promoter, RBS, 5' -UTR and 3' -UTR regions which are necessary for gene transcription and translation and is used as a template for protein synthesis.
2. Preparation of functionalized DNA hydrogel: mu.l dopamine (5% final concentration, 153 molecular weight, pH 7), thiol-modified PCR product, water, 0.4. mu.l four-arm polyethylene glycol thiol (2% final concentration, 20000 average molecular weight, pH 4), 0.4. mu.l four-arm polyethylene glycol maleimide (2% final concentration, 20000 average molecular weight, pH 4), and a total of 10. mu.l system were added to the PCR tube in sequence, each component was well mixed after addition, and the reaction at room temperature allowed a rapid reaction to obtain a porous DNA hydrogel, and the obtained functionalized DNA hydrogel was used for subsequent cell-free protein expression.
3. Cell-free protein expression of functionalized DNA hydrogels
The cell-free expression system comprises the following components: the functional DNA hydrogel containing GFP fluorescent protein gene 100 ng/microliter, 10mM phosphate buffer solution, 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% by volume of Escherichia coli extract are added into a PCR tube, uniformly mixed, shaken under the conditions of 30 ℃ and 1000rpm by using a constant-temperature mixer, taken out after about 12 hours, the expression product is transferred to a black-well plate, and the fluorescence intensity is measured by a microplate reader under the conditions of excitation light 485nm, emission light 535nm and exposure time 0.1s to reflect the expression level of green fluorescent protein. The result shows (fig. 2), under the same condition, compared with the functional DNA hydrogel containing gene template with equal concentration and the common linear template (PCR product), the protein expression level of the functional DNA hydrogel containing 0.8% and 2% of four-arm polyethylene glycol provided by the present patent is significantly better than that of the PCR product as the template.
Test example 1: reuse of functionalized DNA hydrogels
In the CFPS reaction system, a hydrogel containing template DNA, a lysis solution, and an energy buffer are added to complete the expression of fluorescence, and then fresh lysis solution is supplemented to reuse the hydrogel, so that repeated protein production can be achieved, as shown in fig. 3, which shows the reuse of the functionalized DNA hydrogel, and the prepared DNA hydrogel can be reused several tens of times.
Example 2: effect of different concentrations, molecular weight of polymers and reaction time on cell-free protein expression
1. Expression results of DNA hydrogel protein formed by four-arm polyethylene glycol sulfydryl and four-arm polyethylene glycol maleimide with different concentrations and DNA template
The method comprises the steps of mixing uniformly four-arm polyethylene glycol sulfydryl (the final concentration is 0.1%, 0.5%, 0.8%, 1%, 2%, 5%, 25% and 30% respectively) and four-arm polyethylene glycol maleimide (the final concentration is 0.1%, 0.5%, 0.8%, 1%, 2%, 5%, 25% and 30% respectively) with different concentrations, dopamine (the final concentration is 4-6%), a sulfydryl modified PCR product and water, wherein the total 10 mu L of the system is obtained, the porous DNA hydrogel is obtained at room temperature, the expression amounts of hydrogel proteins with different concentrations are shown in figure 4, the concentrations of formed functionalized DNA hydrogel templates are the same, the concentrations of the dopamine are 5%, and the number average molecular weights of the four-arm polyethylene glycol sulfydryl and the four-arm polyethylene glycol maleimide are 20000. Wherein a represents the fluorescence intensity expressed by the functional DNA hydrogel protein formed by 0.5 percent of four-arm polyethylene glycol sulfydryl, 0.5 percent of four-arm polyethylene glycol maleimide and the like; b represents the fluorescence intensity expressed by the functional DNA hydrogel protein formed by 0.8 percent of four-arm polyethylene glycol sulfydryl, 0.8 percent of four-arm polyethylene glycol maleimide and the like; c represents the fluorescence intensity expressed by functional DNA hydrogel protein formed by 1% of four-arm polyethylene glycol sulfydryl, 1% of four-arm polyethylene glycol maleimide and the like; d represents the fluorescence intensity expressed by functional DNA hydrogel protein formed by 2% of four-arm polyethylene glycol sulfydryl, 2% of four-arm polyethylene glycol maleimide and the like; e represents the fluorescence intensity expressed by the functional DNA hydrogel protein formed by 5 percent of four-arm polyethylene glycol sulfydryl, 5 percent of four-arm polyethylene glycol maleimide and the like; f represents the fluorescence intensity expressed by functional DNA hydrogel protein formed by 25 percent of four-arm polyethylene glycol sulfydryl, 25 percent of four-arm polyethylene glycol maleimide and the like; g represents the fluorescence intensity of template protein expression in the solution state.
2. Expression results of DNA hydrogel protein formed by four-arm polyethylene glycol sulfydryl and four-arm polyethylene glycol maleimide with different molecular weights and DNA template
The method comprises the following steps of mixing 10 mu L of four-arm polyethylene glycol sulfydryl (the final concentration is respectively 2%, 5% and 25%, and the number average molecular weight is respectively 1000, 5000 and 20000) and four-arm polyethylene glycol maleimide (the final concentration is respectively 2%, 5% and 25%, and the number average molecular weight is respectively 1000, 5000 and 20000) with different molecular weights, dopamine, a sulfydryl modified PCR product and water in total, uniformly mixing, and obtaining the porous DNA hydrogel at room temperature, wherein hydrogel protein expression amounts formed by polymers with different molecular weights are shown in figure 5, the concentrations of formed functionalized DNA hydrogel templates are the same, and the concentration of the dopamine is 5%. Wherein A represents the fluorescence intensity expressed by the functionalized DNA hydrogel protein formed by the four-arm polyethylene glycol sulfydryl and the four-arm polyethylene glycol maleimide with the number average molecular weight of 5000 and the concentration of 2 percent; b represents the fluorescence intensity expressed by the functionalized DNA hydrogel protein formed by the four-arm polyethylene glycol sulfydryl and the four-arm polyethylene glycol maleimide with the number average molecular weight of 20000 and the concentration of 2 percent; c represents the fluorescence intensity expressed by the functionalized DNA hydrogel protein formed by the four-arm polyethylene glycol sulfydryl and the four-arm polyethylene glycol maleimide with the number average molecular weight of 5000 and the concentration of 5 percent; d represents the fluorescence intensity expressed by the functionalized DNA hydrogel protein formed by the four-arm polyethylene glycol sulfydryl and the four-arm polyethylene glycol maleimide with the number average molecular weight of 20000 and the concentration of 5 percent; e represents the fluorescence intensity expressed by the functionalized DNA hydrogel protein formed by the four-arm polyethylene glycol sulfydryl and the four-arm polyethylene glycol maleimide with the number average molecular weight of 5000 and the concentration of 25 percent; f represents the fluorescence intensity expressed by the functionalized DNA hydrogel protein formed by the four-arm polyethylene glycol sulfydryl and the four-arm polyethylene glycol maleimide with the number average molecular weight of 20000 and the concentration of 25 percent; g represents the fluorescence intensity of template protein expression in the solution state.
3. Influence of different pH values on gelling time of reaction of four-arm polyethylene glycol sulfydryl and four-arm polyethylene glycol maleimide
The porous DNA hydrogel can be obtained by reacting a total of 10. mu.L of four-arm polyethylene glycol sulfydryl (the final concentration is 2%, and the pH values are 7.5, 7, 6, 5, 4 and 3.5) with different pH values, four-arm polyethylene glycol maleimide (the final concentration is 2%, and the pH values are 7.5, 7, 6, 5, 4 and 3.5), dopamine, a sulfydryl modified PCR product and water at room temperature for 1s-12h, as shown in FIG. 6.
Test example 2 determination of mechanical Properties of functionalized DNA hydrogel
Dopamine (final concentration of 5%, molecular weight 153, PH 7), thiol-modified PCR product, water, four-arm polyethylene glycol thiol (final concentration of 2%, 5%, number average molecular weight 20000, PH 4) and four-arm polyethylene glycol maleimide (final concentration of 2%, 5%, number average molecular weight 20000, PH 7) of different concentrations, in total 50 μ L, were added to the PCR tube in sequence, and each component was mixed well and uniformly after addition, and a porous DNA hydrogel could be obtained by reaction. The relationship between the elastic modulus (G ') and the viscous modulus (G') of DNA hydrogels was tested using a rotational rheometer (see FIG. 7), indicating that the polymer is a solid when the elastic modulus is greater than the viscous modulus and vice versa. As shown in FIG. 7, the DNA hydrogel has G 'always larger than G' in the tested frequency range, and shows good hydrogel elastic mechanical properties.
In conclusion, by the technical scheme of the invention, the preparation process of the functionalized DNA hydrogel provided by the invention is simple, rapid and low in cost, does not need conditions such as initiation and procoagulant, has a rapid gelling speed in preparation, is generally within 60 seconds, has high feasibility of implementation, and can be used for cell-free protein expression; meanwhile, the functional DNA hydrogel has high mechanical strength and is not easy to break, so that a sulfhydryl-modified PCR product can be protected, the local effective concentration of a template can be increased, and the protein expression amount is further improved; and the hydrogel can be repeatedly used, so that the protein expression cost is reduced.
The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only 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 invention.
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 disclosure.
Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.
Unless specifically stated otherwise, use of the terms "comprising", "including", "having" or "having" is generally to be understood as 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 referents unless the context clearly dictates otherwise. In addition, where the term "about" is used before a quantity, the present teachings also include the particular quantity itself unless specifically stated otherwise.
It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
In addition, the inventors of the present invention have also made experiments with other materials, process operations, and process conditions described in the present specification with reference to the above examples, and have obtained preferable results.
While the invention has been described with reference to illustrative embodiments, 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 invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention 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 (10)
1. A functionalized DNA hydrogel is characterized in that the functionalized DNA hydrogel is prepared by cross-linking reaction of a macromolecular compound containing maleimide groups, a macromolecular compound containing sulfydryl groups, dopamine and DNA modified by sulfydryl groups.
2. The functionalized DNA hydrogel of claim 1, wherein: the maleimide group-containing polymer compound or thiol group-containing polymer compound is multi-armed, preferably four-armed or eight-armed, and particularly preferably four-armed;
preferably, the maleimide group-containing polymer compound comprises a four-arm polyethylene glycol maleimide; preferably, the average molecular weight of the four-arm polyethylene glycol maleimide is 1000-20000 g/mol;
preferably, the thiol-group-containing polymer compound includes a four-arm polyethylene glycol thiol group; preferably, the average molecular weight of the sulfhydryl of the four-arm polyethylene glycol is 1000-20000 g/mol.
3. The functionalized DNA hydrogel of claim 1, wherein: the elastic modulus of the functionalized DNA hydrogel is 300-1200 Pa within the frequency range of 0.1-5 Hz.
4. A method for preparing a functionalized DNA hydrogel, which is characterized by comprising the following steps:
and carrying out crosslinking reaction on a uniformly mixed system containing a maleimide-containing macromolecular compound, a thiol-containing macromolecular compound, dopamine and thiol-modified DNA at room temperature to obtain the functionalized DNA hydrogel.
5. The method of claim 4, wherein: the crosslinking reaction comprises an addition reaction and a polymerization reaction; and/or the time of the crosslinking reaction is 1s to 12h, preferably 60s or less.
6. The method of claim 4, wherein: the maleimide group-containing polymer compound or thiol group-containing polymer compound is multi-armed, preferably four-armed or eight-armed, and particularly preferably four-armed;
preferably, the maleimide group-containing polymer compound comprises a four-arm polyethylene glycol maleimide; preferably, the average molecular weight of the four-arm polyethylene glycol maleimide is 1000-20000 g/mol; preferably, the concentration of the polymer compound containing maleimide groups in the uniform mixing system is 0.1-30 wt%; preferably, the pH value of the polymer compound containing maleimide groups is 3.5-7.5;
preferably, the thiol-group-containing polymer compound includes a four-arm polyethylene glycol thiol group; preferably, the average molecular weight of the sulfhydryl of the four-arm polyethylene glycol is 1000-20000 g/mol; preferably, the concentration of the thiol-containing macromolecular compound in the uniform mixing system is 0.1-30 wt%; preferably, the pH value of the thiol-group-containing macromolecular compound is 3.5-7.5;
preferably, the concentration of the sulfhydryl-modified DNA in the uniform mixing system is 5-50 ng/muL;
preferably, the concentration of dopamine in the uniformly mixed system is 4-6 wt%.
7. A functionalized DNA hydrogel prepared by the method of any one of claims 4 to 6.
8. Use of the functionalized DNA hydrogel of any one of claims 1-3, 7 in the field of cell-free protein expression.
9. A method of synthesizing a protein, comprising:
providing a functionalized DNA hydrogel according to any one of claims 1-3, 7;
and synthesizing protein by using the functionalized DNA hydrogel.
10. The method according to claim 9, characterized in that it comprises in particular: mixing a cell extract, an energy buffer solution and hydrogel according to a volume ratio of (10-100): (10-100): (1-50), and shaking the obtained mixed solution for 0.5-48h at 0-37 ℃ and a rotation speed of 0-2000rpm to obtain a solution containing the expressed protein, so as to synthesize the protein;
preferably, the cell extract is derived from a prokaryotic cell or a eukaryotic cell;
preferably, the energy buffer solution comprises an amino acid mixed solution, a reaction buffer solution and an energy supplementing solution;
particularly preferably, the amino acid mixture comprises a mixture of leucine, isoleucine, methionine, tyrosine, glycine, alanine, valine, serine, threonine, hemisarcosine, asparagines, phenylalanine, tryptophan, aspartic acid, glutamic acid, lysine, arginine and histidine; particularly preferably, the molar concentration of the amino acid mixed solution is 1-4 mmol/L;
particularly preferably, the reaction buffer solution comprises 1-3% of polyethylene glycol 8000, 1-3 mmol/L of dithiothreitol, 60-80 mmol/L of potassium glutamate, 3-6 mmol/L of magnesium glutamate and 14-18 mmol/L of sucrose, and the solvent is deionized water;
particularly preferably, the energy supplementing liquid comprises 0.8-1 mmol/L guanine-5 ' -triphosphate, 1-2 mmol/L adenosine 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-phosphoglyceride, and the solvent is deionized water.
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| CN111040198A (en) * | 2019-12-30 | 2020-04-21 | 苏州珀罗汀生物技术有限公司 | DNA hydrogel, preparation method and application thereof |
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| CN107569448A (en) * | 2017-09-06 | 2018-01-12 | 青岛大学 | A kind of preparation method and applications of Self-assembled DNA hydrogel |
| CN109810265A (en) * | 2018-12-28 | 2019-05-28 | 天津大学 | A kind of the DNA- polysaccharide hybridized hydrogel and preparation method of solvent driving volume change |
| CN111620985A (en) * | 2019-02-28 | 2020-09-04 | 康码（上海）生物科技有限公司 | Hydrogel for covalent fixation of DNA and application thereof |
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