Preparation method of DNA hydrogel

Abstract

The invention relates to the technical field of hydrogel preparation, and discloses a preparation method of DNA hydrogel, which comprises the following steps: a, using plasmid as a template, and melting double strands of the plasmid template through alkaline denaturation; b, adding a random primer, enabling the plasmid template to be combined with the random primer in a renaturation process, and producing a large amount of double-stranded DNA with an ultra-long chain length and a high-branch topological structure as a DNA hydrogel precursor through hyper-branched rolling circle replication amplification; c, adding a dehydrating agent into the DNA hydrogel precursor solution, and realizing self-assembly by utilizing dense physical entanglement among double-stranded DNA molecules under the induction of the dehydrating agent to form a DNA aggregate; and d, collecting the DNA aggregate, drying the DNA aggregate to remove the residual dehydrating agent, resuspending the DNA aggregate in a buffer solution to enable the DNA aggregate to absorb water and swell, and finally forming the DNA hydrogel with a stable structure. According to the invention, the design and preparation process of the DNA hydrogel can be greatly simplified, the availability of the DNA hydrogel is improved, and the transformation of the DNA hydrogel from basic research to practical application is promoted.

Description

Technical Field

[0001] This invention relates to the field of hydrogel preparation technology, and in particular to a method for preparing a DNA hydrogel. Background Technology

[0002] DNA hydrogels are a new type of three-dimensional network polymer material composed mainly of DNA. They have good biocompatibility, degradability and sequence programmability, and show broad application prospects in drug delivery, tissue engineering, organoid culture and biosensing.

[0003] The rolling circle amplification (RCA) strategy is one of the mainstream methods for preparing pure DNA hydrogels. This method uses artificially designed circular single-stranded DNA as a template, and isothermally amplifies it under the action of Phi29 DNA polymerase to generate ultralong single-stranded DNA molecules containing periodically repeating sequences. These molecules then self-assemble into DNA hydrogels through interstrand base hybridization and physical entanglement. However, this preparation method has several inherent drawbacks in practical applications, which restrict the large-scale preparation and widespread application of DNA hydrogels.

[0004] First, the preparation process of circular single-stranded DNA templates is cumbersome and technically demanding. Linear DNA must be pre-circulated into a single-stranded circular template, a process involving multiple steps such as sequence design, chemical synthesis, and enzymatic ligation and circularization, which is complex and time-consuming. Due to limitations in conventional chemical synthesis processes, the length of circular single-stranded DNA templates is typically limited to 150 bases, making it difficult to incorporate long functional elements such as gene coding sequences. Furthermore, the preparation efficiency of circular single-stranded DNA is significantly affected by the secondary structure of the sequence; some sequences with complex secondary structures are difficult to circularize efficiently, leading to a high risk of template construction failure and low preparation efficiency, further increasing the cost of large-scale applications.

[0005] Secondly, traditional RCA preparation methods based on single-stranded circular templates mainly produce ultra-long single-stranded DNA, with interstrand crosslinking primarily through localized base pairing and weak physical entanglement, resulting in a relatively low network crosslinking density. Studies have shown that at high concentrations, these single-stranded products readily form tight DNA nanoflower structures through intrastrand folding, which in turn inhibits interstrand entanglement, preventing the formation of stable macroscopic hydrogels (see physical examples). Figure 1To overcome this deficiency, researchers have attempted various improvement strategies: Yao et al. introduced a dual RCA strategy, generating partially complementary ultra-long single chains through two sets of RCA reactions, which, after mixing, rapidly form a physical cross-linking network to improve gel stability; Xu et al. proposed a method of cold-treating cyclic templates to enhance subsequent inter-chain entanglement by inducing slight template aggregation. However, these improvements have failed to break free from the technical framework of relying on cyclic single-chain templates and inter-chain base hybridization for gelation, and core problems such as cumbersome template construction, limited sequence design, and unstable gelation remain unresolved. Summary of the Invention

[0006] In order to overcome the shortcomings of existing technologies that rely on cyclic single-strand templates and inter-strand base hybridization for gelation, such as cumbersome template construction, limited sequence design, and unstable gelation.

[0007] The present invention adopts the following technical solution: A method for preparing a DNA hydrogel includes the following steps: a. Using a plasmid as a template, the double strand of the plasmid template is destranded by alkaline denaturation, wherein the plasmid is a circular double-stranded DNA; b. Add random primers so that the plasmid template binds to the random primers through complementary base pairing during renaturation. Then, superbranched rolling circle replication and amplification are performed to generate a large amount of double-stranded DNA with ultra-long chain length and high branching topology, which serves as a DNA hydrogel precursor. c. Add a dehydrating agent to the DNA hydrogel precursor solution. Under the induction of the dehydrating agent, DNA molecules become densely physically entangled and aggregate to form DNA aggregates. d. The DNA aggregates are collected by centrifugation, dried to remove residual dehydrating agent, and then resuspended in buffer solution to allow them to absorb water and swell, ultimately forming a structurally stable DNA hydrogel.

[0008] The reaction mechanism of this invention is as follows: A superbranched rolling circle replication amplification reaction generates double-stranded DNA with ultra-long chain length and a highly branched topology, providing a structural basis for subsequent hydrogel network formation. A dehydrating agent significantly reduces the solubility of DNA in a mixed solvent by disrupting the hydration layer on the surface of DNA molecules, inducing phase separation. During this process, the local DNA concentration increases sharply, causing dense physical entanglement between the ultra-long double-stranded DNA molecules with highly branched topology, forming DNA aggregates. After centrifugation and drying to remove the dehydrating agent, the aggregates are resuspended in a buffer solution. Water molecules re-embed into the physical entanglement network between DNA molecules, further stabilizing the three-dimensional structure, thereby obtaining a structurally stable DNA hydrogel.

[0009] Optionally, the dehydrating agent may be one or more of ethanol and isopropanol.

[0010] Preferably, the dehydrating agent is ethanol, and the DNA hydrogel precursor solution is mixed with ethanol at a volume ratio of 1:(2-3).

[0011] Optionally, the random primer is a random hexamer.

[0012] Optionally, the reactants in step b are incubated at 30-37°C and 300-600 rpm for 12-16 hours.

[0013] Preferably, the alkaline denaturation process uses a NaOH solution with a concentration of 0.1M to 0.2M.

[0014] Optionally, polymerase can be added in step b to achieve rolling circle replication and amplification.

[0015] Optionally, the polymerase used is phi29 DNA polymerase.

[0016] Optionally, phi29 DNA polymerase, phi29 DNA polymerase buffer, and dNTPs may be added in step b.

[0017] Preferably, the reactants obtained in step c must reach a viscous state before being collected by centrifugation.

[0018] Preferably, the concentration of dNTPs is 1-2 mM.

[0019] The beneficial effects of the present invention include at least the following: 1) In existing research on DNA hydrogel preparation using RCA technology, the general approach follows the technical path of "preparing a circular single-stranded template, using specific primers to initiate RCA to generate ultra-long single-stranded DNA, and relying on single-stranded DNA inter-strand hybridization and physical entanglement to form a gel." Unlike traditional methods that focus on optimizing the circular single-stranded template and enhancing inter-strand hybridization of single-stranded DNA, this invention breaks away from the existing technical framework and proposes a completely new technical route: ① Template innovation: Directly using any double-stranded plasmid as the starting template, it undergoes simple alkaline denaturation to partially unwind, eliminating the cumbersome steps of chemical synthesis and enzymatic cyclization of circular single-stranded templates in existing technologies; ② Product structure innovation: Under the drive of random primers, superbranched rolling circle replication is performed, generating double-stranded DNA with ultra-long strand length and highly branched topology in a one-step process. This double-stranded structure avoids the intrastrand folding that easily occurs in single-stranded DNA, fundamentally solving the technical problem of unstable gelation caused by intrastrand self-assembly of RCA single-stranded products in traditional technical approaches; ③ Innovative gelation mechanism: Dehydration-induced physical entanglement is used for gelation. Utilizing the synergistic effect of the aforementioned highly branched topology and dehydration induction, dense, stable, and non-specific physical entanglements can be formed between double-stranded DNA, thus forming a three-dimensional hydrogel network. This is fundamentally different from the gelation methods of existing technical approaches that highly rely on interstrand base hybridization. To enhance interstrand hybridization, existing technologies rely on specific sequence design of templates and primers. This invention eliminates the dependence on sequence design and hybridization, and has greater versatility.

[0020] 2) This invention can directly use any plasmid as a starting template, eliminating the need for pre-preparation of circular single-stranded DNA templates through chemical synthesis and enzymatic cyclization, as is required in traditional methods. This improvement not only eliminates the cumbersome template construction steps and greatly simplifies the preparation process, but also allows for flexible introduction of long functional elements such as gene coding sequences, regardless of template sequence length or secondary structure. This significantly improves the preparation efficiency of DNA hydrogels and reduces the technical threshold and preparation cost.

[0021] 3) This invention generates double-stranded DNA with ultra-long chain length and highly branched topology in a one-step process through random primer-driven superbranched rolling circle replication. This double-stranded product exhibits a homogeneous viscous state in solution without forming a precipitate. This fundamentally overcomes the inherent defects of traditional RCA single-stranded products, which easily form DNA nanoflowers through intra-strand self-assembly, leading to unstable gelation and uneven network. This ensures the homogeneity of the hydrogel precursor solution and the controllability of the subsequent gelation process.

[0022] 4) The gelation mechanism of traditional RCA hydrogels is highly dependent on base complementarity hybridization between single-stranded DNA products, requiring careful sequence design to achieve interstrand crosslinking. This invention achieves pure topological entanglement gelation independent of sequence hybridization through the synergistic effect of highly branched double-stranded DNA topology and dehydration induction, eliminating the dependence on template and primer specific sequence design, and improving the versatility of the method and the availability of DNA hydrogels. Attached Figure Description

[0023] Figure 1 This is a photograph of the product obtained by the traditional RCA preparation method based on a single-chain cyclic template. Figure 2 This is a reaction mechanism diagram of a method for preparing a DNA hydrogel according to the present invention; Figure 3 This is a physical image of step b in embodiment 1 of the present invention; Figure 4 This is a photograph of the hydrogel precursor obtained in step b of Embodiment 1 of the present invention. Figure 5 This is a physical image of step c in embodiment 1 of the present invention; Figure 6 This is a photograph of the hydrogel finally obtained in step d of embodiment 1 of the present invention. Figure 7 This is a scanning electron microscope image of the DNA hydrogel prepared in this invention; Figure 8 These are rheological test images of the DNA hydrogel prepared in this invention; Figure 9 These are images of the DNA hydrogel prepared according to the present invention for biocompatibility testing. Detailed Implementation

[0024] The present invention will now be further described with reference to the accompanying drawings and specific embodiments.

[0025] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0026] Unless otherwise specified, the raw materials and equipment used in this invention are all conventional raw materials and equipment in the art and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in this invention are all conventional methods in the art.

[0027] As used in this invention, unless otherwise clearly stated, the singular forms “a,” “an,” and “the” include embodiments with plural indicators.

[0028] As used in the specification and appended claims of this invention, unless otherwise clearly stated, the term "or" is generally used in its meaning as including "and / or".

[0029] Unless otherwise indicated, all figures expressing characteristic magnitudes, quantities, and physical properties will be understood to be modified by the term “about”, regardless of whether the term “about” is immediately present. Therefore, unless indicated to the contrary, the numerical parameters presented are approximations that may vary depending on the desired properties sought by those skilled in the art using the teachings disclosed herein.

[0030] In this invention, the phi29 DNA polymerase used is a product from Thermo Fisher Scientific's China website.

[0031] In this invention, the random hexamer is NNNNNN, where N is any base in A / T / C / G.

[0032] In this invention, BV2 cells are immortalized cell lines obtained from mouse microglia. Specifically, products from Wuhan Shangen Biotechnology Co., Ltd. can be used.

[0033] In this invention, dNTP, an abbreviation for deoxyribonucleoside triphosphate, is a collective term including dATP, dGTP, dTTP, dCTP, etc. N refers to a nitrogenous base, and the variable represents one of A, T, G, C, etc.

[0034] In this invention, PBS (phosphate buffer saline) is a commonly used buffer solution in biological experiments. It is prepared by mixing disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), sodium chloride (NaCl), and other components in a specific ratio. The pH value is usually maintained at 7.2-7.4, and the osmotic pressure is similar to that of human body fluids.

[0035] The rheometer from Mettler Toledo GmbH, Germany, can be used in this invention.

[0036] The Calcein-AM / PI reagent in this invention is a reagent combination widely used in cell biology and medical research, mainly for distinguishing between viable and dead cells.

[0037] In this invention, the Calcein-AM / PI reagent can be the Calcein-AM / PI live / dead cell double staining kit produced by Beijing Solarbio Science & Technology Co., Ltd.

[0038] like Figure 2 As shown, a method for preparing a DNA hydrogel includes the following steps: a. Using a plasmid as a template, the double strand of the plasmid template is destranded by alkaline denaturation, wherein the plasmid is a circular double-stranded DNA; b. Add random primers so that the plasmid template can bind to the random primers through complementary base pairing during the renaturation process. Add polymerase to achieve superbranched rolling circle replication and amplification, and produce a large amount of double-stranded DNA with ultra-long chain length and high branching topology as DNA hydrogel precursor; c. Add a dehydrating agent to the DNA hydrogel precursor solution. Under the induction of the dehydrating agent, the DNA molecules achieve self-assembly by the dense physical entanglement between the double-stranded DNA molecules, forming DNA aggregates. d. After drying the obtained DNA aggregates, they were resuspended in PBS solution. The DNA aggregates absorbed water, swelled, and formed a DNA hydrogel structure.

[0039] In some embodiments, the dehydrating agent is one or more of ethanol and isopropanol.

[0040] In some embodiments, the random primer is a random hexamer.

[0041] In some embodiments, the polymerase used is phi29 DNA polymerase. In step b, phi29 DNA polymerase, phi29 DNA polymerase buffer, and dNTPs are added.

[0042] In some embodiments, the final concentration of dNTPs is 1-2 mM.

[0043] In some embodiments, the reactants in step b are incubated at 30-37°C and 300-600 rpm for 12-16 hours.

[0044] In some embodiments, the amount of plasmid used is 1-10 μg.

[0045] In some embodiments, the DNA hydrogel precursor solution and the dehydrating agent are mixed at a volume ratio of 1:(2-3).

[0046] In some embodiments, the alkaline denaturation process uses a NaOH solution with a concentration of 0.1 M to 0.2 M. Other alkaline solutions may also be used.

[0047] In some embodiments, the reactants obtained in step c need to reach a viscous state before being collected by centrifugation.

[0048] Specific implementation examples are shown in Examples 1, 2 and 3.

[0049] Example 1: This invention discloses a method for preparing a DNA hydrogel, comprising the following steps: Step a. Dilute the puc57 plasmid obtained through gene synthesis to 1 μg / μL with sterile water. Take 10 μg of plasmid, add 50 μL of 0.1M NaOH solution, add sterile water to make up to 100 μL, and incubate at room temperature for 5 min to denature the double-stranded plasmid DNA.

[0050] Step b. Using random hexamer as the rolling circle replication amplification primer, add 250 μL of random hexamer primer (10 μM) to the above reaction system to allow the primer to bind to the plasmid template and obtain the reaction solution.

[0051] like Figure 3 As shown, in the above reaction system, 20 μL of phi29 DNA polymerase (10 U / μL, 200 U used), 50 μL of 10×phi29 DNA polymerase buffer, and 50 μL of 10 mM dNTP were added to 350 μL of reaction solution. The volume was then brought to 500 μL with sterile water, and the mixture was gently mixed by pipetting or inverting several times. After preparation, the reaction system was placed in a constant temperature shaker and incubated at 30°C and 300 rpm for 12 hours. When the solution reached a high-viscosity, stringy state, it indicated that the superbranched rolling circle replication amplification reaction had been successfully completed, yielding a DNA hydrogel precursor solution composed of ultra-long, highly branched double-stranded DNA (e.g., ...). Figure 4 (As shown).

[0052] Step c. Mix the DNA hydrogel precursor solution with ethanol at a 1:2 ratio. DNA molecules will rapidly aggregate, forming white aggregates. Centrifuge the sample at 4°C and 12000 rpm for 20 min, remove the supernatant, and obtain the white DNA aggregates (e.g., ...). Figure 5 (As shown), invert the centrifuge tube or leave it open at room temperature for about 20 minutes to allow the residual ethanol in the precipitate to evaporate fully.

[0053] Step d. Add 200 μL of PBS solution to the DNA aggregates. The DNA aggregates gradually swell and eventually form a stable DNA hydrogel (e.g., ...). Figure 6 (As shown).

[0054] Example 2: This invention discloses a method for preparing a DNA hydrogel, comprising the following steps: Step a. Dilute the puc57 plasmid obtained through gene synthesis to 1 μg / μL with sterile water. Take 1 μg of plasmid, add 50 μL of 0.2M NaOH solution, add sterile water to make up to 100 μL, and incubate at room temperature for 3 min to denature the double-stranded plasmid DNA.

[0055] Step b. Using random hexamer as rolling circle replication amplification primers, add 250 μL of random hexamer primers (20 μM) to the above reaction system. The primers bind to the plasmid template, and the primers anneal to the single-stranded regions of the template to obtain the reaction solution.

[0056] In the above reaction system, 20 μL of 10 U / μL phi29 DNA polymerase, 50 μL of 10×phi29 DNA polymerase buffer, and 50 μL of 20 mM dNTP were added to 350 μL of reaction solution. The volume was then brought to 500 μL with sterile water, and the mixture was gently mixed by pipetting or inverting several times. After preparation, the reaction system was placed in a constant temperature shaker and incubated at 37°C and 600 rpm for 16 h. When the solution reached a high-viscosity, stringy state, it indicated that the superbranched rolling circle replication amplification reaction had been successfully completed, yielding a DNA hydrogel precursor solution composed of ultra-long, highly branched double-stranded DNA.

[0057] Step c. Mix the DNA hydrogel precursor solution with ethanol at a ratio of 1:3. DNA molecules will rapidly aggregate, forming white aggregates. Centrifuge the sample at 4°C and 12000 rpm for 10 minutes. Remove the supernatant to obtain white DNA aggregates. Invert the centrifuge tube or leave it open at room temperature for approximately 20 minutes to allow residual ethanol in the precipitate to evaporate completely.

[0058] Step d. Add 200 μL of PBS solution to the DNA aggregates. The DNA aggregates gradually swell and eventually form a stable DNA hydrogel.

[0059] Example 3: This invention discloses a method for preparing a DNA hydrogel, comprising the following steps: Step a. Dilute the puc57 plasmid obtained through gene synthesis to 1 μg / μL with sterile water. Take 1 μg of plasmid, add 50 μL of 0.1M NaOH solution, add sterile water to make up to 100 μL, and incubate at room temperature for 5 min to denature the double-stranded plasmid DNA.

[0060] Step b. Using random hexamer as rolling circle replication amplification primers, add 250 μL of random hexamer primers (20 μM) to the above reaction system. The primers bind to the plasmid template, and the primers anneal to the single-stranded regions of the template to obtain the reaction solution.

[0061] In the above reaction system, 20 μL of 10 U / μL phi29 DNA polymerase, 50 μL of 10×phi29 DNA polymerase buffer, and 50 μL of 20 mM dNTP were added to 350 μL of reaction solution. The volume was then brought to 500 μL with sterile water, and the mixture was gently pipetted or inverted several times to mix. After preparation, the reaction system was placed in a constant temperature shaker and incubated at 37°C and 600 rpm for 16 h. When the solution reached a high-viscosity, stringy state, it indicated that the superbranched rolling circle replication amplification reaction had been successfully completed, yielding a DNA hydrogel precursor solution composed of ultra-long, highly branched double-stranded DNA.

[0062] Step c. Mix the DNA hydrogel precursor solution with isopropanol at a ratio of 1:0.6. DNA molecules will rapidly aggregate, forming white aggregates. Centrifuge the sample at 4°C and 12000 rpm for 10 minutes. Remove the supernatant to obtain white DNA aggregates. Invert the centrifuge tube or leave it open at room temperature for approximately 20 minutes to allow any residual isopropanol in the precipitate to evaporate completely.

[0063] Step d. Add 200 μL of PBS solution to the DNA aggregates. The DNA aggregates gradually swell and eventually form a stable DNA hydrogel.

[0064] The DNA hydrogel samples prepared in Example 1 were characterized by scanning electron microscopy, rheological properties, and biocompatibility.

[0065] (1) The internal structure of the DNA hydrogel was characterized using scanning electron microscopy: Take 100 μL of the DNA hydrogel sample from Example 1, rapidly freeze it in liquid nitrogen, cut it, and then dry it in a vacuum freeze dryer for 24 hours. Adhere the sample section to the sample stage, sputter-coat it with gold, and use a scanning electron microscope to image and observe the sample. Figure 7 As can be seen, the DNA hydrogel exhibits a loose and porous three-dimensional network structure inside.

[0066] (2) The rheological properties of the DNA hydrogel were characterized using a rheometer: A parallel plate fixture with a diameter of 8 mm was selected, and the test gap was set to 1 mm. Dynamic time-scan tests were performed at 25°C, with a fixed strain of 2% and a fixed frequency of 0.2 Hz to monitor the changes in the storage modulus (G') and loss modulus (G'') of the hydrogel samples over time. Figure 8 As can be seen, the storage modulus (G') of the DNA hydrogel is always significantly higher than the loss modulus (G"), and the two remain basically stable, indicating that the DNA hydrogel forms a structurally stable three-dimensional network and exhibits typical elastic gel characteristics.

[0067] (3) DNA hydrogel biocompatibility test: BV2 cells were co-incubated with DNA hydrogel for 48 hours, with BV2 cells without DNA hydrogel serving as a blank control. After incubation, BV2 cells were stained with Calcein-AM / PI reagent, and cell viability was observed under a fluorescence microscope. Figure 9 As shown, similar to the blank control group, BV2 cells co-incubated with the DNA hydrogel all exhibited bright green fluorescence (live cells), with only a very small amount of red fluorescence (dead cells), indicating that cell survival and proliferation were not significantly affected. These results demonstrate that the DNA hydrogel prepared by this method has good biocompatibility.

[0068] The above description is merely a preferred embodiment of the present invention and does not limit the scope of patent protection of the present invention. Any equivalent structural transformations made based on the description and drawings of the present invention, whether directly or indirectly applied to other related technical fields, are similarly included within the scope of protection of the present invention.

Claims

1. A method for preparing a DNA hydrogel, characterized in that, Includes the following steps: a. Using a plasmid as a template, the double strand of the plasmid template is destranded by alkaline denaturation, wherein the plasmid is a circular double-stranded DNA; b. Add random primers so that the plasmid template binds to the random primers through complementary base pairing during renaturation. Then, perform superbranched rolling circle replication amplification to produce a large amount of double-stranded DNA with ultra-long chain length and highly branched topology, which serves as a DNA hydrogel precursor. c. Add a dehydrating agent to the DNA hydrogel precursor solution. Under the induction of the dehydrating agent, DNA molecules become densely physically entangled and aggregate to form DNA aggregates. d. Collect DNA aggregates, dry them to remove residual dehydrating agent, and then resuspend them in buffer solution to allow them to absorb water and swell, eventually forming a structurally stable DNA hydrogel; the dehydrating agent is ethanol, and the DNA hydrogel precursor solution is mixed with ethanol in a ratio of 1:(2-3).

2. The method for preparing the DNA hydrogel as described in claim 1, characterized in that, The random primers are random hexamers.

3. The method for preparing the DNA hydrogel as described in claim 1, characterized in that, In step b, the reactants are incubated at 30-37°C and 300-600 rpm for 12-16 hours.

4. The method for preparing the DNA hydrogel according to any one of claims 1 to 3, characterized in that, In step b, polymerase is added to achieve rolling circle replication and amplification.

5. The method for preparing the DNA hydrogel as described in claim 4, characterized in that, The polymerase used is phi29 DNA polymerase.

6. The method for preparing the DNA hydrogel as described in claim 5, characterized in that, In step b, add phi29 DNA polymerase, phi29 DNA polymerase buffer, and dNTPs.

7. The method for preparing the DNA hydrogel according to any one of claims 1 to 3, characterized in that, The reactants obtained in step c must reach a viscous state before centrifugation and collection.

8. The method for preparing the DNA hydrogel according to any one of claims 1 to 3, characterized in that, The alkaline denaturation process uses NaOH solution with a concentration of 0.1M to 0.2M.

Images