WO2022218302A1 - 一种力学性能提高的核酸水凝胶及其制备方法和用途 - Google Patents

一种力学性能提高的核酸水凝胶及其制备方法和用途 Download PDF

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WO2022218302A1
WO2022218302A1 PCT/CN2022/086336 CN2022086336W WO2022218302A1 WO 2022218302 A1 WO2022218302 A1 WO 2022218302A1 CN 2022086336 W CN2022086336 W CN 2022086336W WO 2022218302 A1 WO2022218302 A1 WO 2022218302A1
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nucleic acid
cross
acid strand
scaffold
unit
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WO2022218302A9 (zh
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刘冬生
李宇杰
杨勃
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清华大学
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/0052—Preparation of gels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/0052—Preparation of gels
    • B01J13/0065—Preparation of gels containing an organic phase

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  • the present disclosure belongs to the field of polymer materials and biotechnology, and in particular, the present disclosure relates to a nucleic acid hydrogel and a preparation method and use thereof.
  • Supramolecular hydrogel is a kind of soft matter material with three-dimensional network structure formed by self-assembly based on non-covalent interaction.
  • the transient reversibility of non-covalent interactions endows supramolecular hydrogels with favorable dynamic properties, such as stimuli responsiveness, injectability, shape adaptability, and Self-healing properties, etc., show great application potential in many fields such as biomedicine, flexible electronic materials, and soft robots.
  • the fast dissociation and recombination rates of supramolecular interactions result in poor mechanical properties of supramolecular hydrogels, which limit the application of supramolecular hydrogels as structural materials in many fields. Therefore, how to design and fabricate a hydrogel with high mechanical strength and dynamic properties is a long-term challenge.
  • Nanomaterials such as carbon nanoparticles, quantum dots, metal nanoparticles, etc.
  • supramolecular hydrogels can also be introduced into supramolecular hydrogels as additional cross-linking points, which can improve the mechanical properties of hydrogels while also imparting supramolecular hydrogels. Additional physical properties of glue. But the introduction of nanomaterials may bring unknown biological toxicity.
  • these methods will change the crosslinking structure and crosslinking density of the hydrogel network while enhancing the mechanical properties, and the original topology, mesh distribution and permeability of the supramolecular hydrogel network will no longer be preserved.
  • Nucleic acid molecules can encode, store and transmit genetic information, and are one of the core molecules of living systems.
  • people have not only carried out a lot of research and exploration on nucleic acid molecules as carriers of genetic information, but also began to use them as assembly materials.
  • nucleic acid molecules have the characteristics of clear structure, specific recognition of bases, and easy functional modification. They have received extensive attention and development in the fields of chemistry, biology, and materials science.
  • people have designed and synthesized a variety of nucleic acid nanomaterials by taking advantage of the excellent properties of nucleic acid molecules.
  • nucleic acid hydrogel is a typical representative.
  • nucleic acid molecules to make hydrogels can utilize both the skeleton function of the hydrogel and the biological function of DNA to achieve the unification of the structure and function of the hydrogel material. Fusion has a wide range of applications in multiple biomedical fields including drug delivery and sustained release, biological detection, protein production, and immune regulation.
  • Nucleic acid hydrogels have broad application prospects in many fields. How to further improve the mechanical properties of nucleic acid hydrogels and make nucleic acid hydrogels have the dynamic characteristics of supramolecular hydrogels while improving their mechanical strength is a topic in the art. important issues that need to be addressed urgently.
  • nucleic acid hydrogel which is formed by cross-linking a scaffold unit with a cross-linking unit I and a cross-linking unit II, respectively.
  • a nucleic acid hydrogel which is formed by cross-linking a scaffold unit with a cross-linking unit I and a cross-linking unit II, respectively.
  • the present disclosure first provides a nucleic acid hydrogel, wherein the nucleotide hydrogel comprises a scaffold unit, and a cross-linking unit I and a cross-linking unit II respectively cross-linked with the scaffold unit;
  • the scaffold unit includes a scaffold core and at least 3 first nucleic acid strands combined with the scaffold core, and the cross-linking unit 1 includes a cross-linked core and at least 2 second nucleic acid strands combined with the cross-linked core;
  • One end of the first nucleic acid strand away from the scaffold core is a sticky end, and is complementary to the sequence of the second nucleic acid strand away from the sticky end of the cross-linked core;
  • the cross-linking unit II includes at least two repeating segments, the repeating segments include a single-stranded third nucleic acid strand and a double-stranded fourth nucleic acid strand, and the cross-linking unit II is composed of the third nucleic acid strand and the double-stranded fourth nucleic acid strand.
  • the fourth nucleic acid strand is alternately connected, and the third nucleic acid strand is complementary to the sequence of the cohesive end of the first nucleic acid strand.
  • the content of the repeating fragments is 0.5 based on the total moles of repeating fragments in the cross-linking unit I and the cross-linking unit II. -99%; optionally, the content of the repeating segment is 1-20%; preferably, the content of the repeating segment is 2.5-10%.
  • the nucleic acid hydrogel according to the present disclosure wherein the materials forming the scaffold core or the cross-linked core are independently selected from the group consisting of nucleic acids, polypeptides, polymeric compounds and nanoparticles.
  • nucleic acid hydrogel according to the present disclosure wherein any core of any one of the first nucleic acid strand, the second nucleic acid strand, the third nucleic acid strand, and the fourth nucleic acid strand nucleotides are modified nucleotides or unmodified nucleotides;
  • any nucleotide of any one of the first nucleic acid strand, the second nucleic acid strand, the third nucleic acid strand and the fourth nucleic acid strand is a deoxyribonucleotide or a ribonucleotide.
  • the length of the sticky end of any one of the first nucleic acid strand and the second nucleic acid strand is 4 nt or more, preferably 4-30 nt, More preferably 4-20nt;
  • the length of the third nucleic acid strand is 4 nt or more, preferably 4-30 nt, more preferably 4-20 nt.
  • the length of the fourth nucleic acid strand is 4 nt or more, preferably 10-40 nt, more preferably 20-30 nt.
  • the present disclosure also provides a method for preparing the nucleic acid hydrogel according to the present disclosure, wherein the method includes the step of cross-linking the scaffold unit with the cross-linking unit I and the cross-linking unit II.
  • the method according to the present disclosure wherein the method comprises the steps of:
  • cross-linking unit I and cross-linking unit II in a gel matrix to obtain a cross-linking unit solution
  • the scaffold unit solution is mixed with the cross-linking unit solution, and the scaffold unit is self-assembled with the cross-linking unit I and the cross-linking unit II to obtain a nucleic acid hydrogel.
  • the method according to the present disclosure wherein the step of preparing the cross-linking unit II comprises:
  • the long-chain single-stranded nucleic acid chain comprising at least 2 single-stranded regions N1 and at least 2 single-stranded regions N2;
  • the long-chain single-stranded nucleic acid strand and the complementary strand are mixed in a gel matrix and subjected to annealing treatment, and the complementary strand is complementary to the sequence of the single-stranded region N1 to form a double-stranded fourth nucleic acid strand.
  • Region N2 forms the third nucleic acid chain, and obtains the cross-linking unit II in which the fourth nucleic acid chain and the third nucleic acid chain are alternately connected;
  • a long-chain single-stranded nucleic acid chain is prepared;
  • long single-stranded nucleic acid strands are prepared in the presence of pyrophosphatase.
  • the present disclosure also provides a method for preparing a nucleic acid hydrogel, comprising the following steps:
  • the scaffold unit comprising a scaffold core and at least 3 first nucleic acid strands bound to the scaffold core;
  • the cross-linking unit I comprising a cross-linking core and at least 2 second nucleic acid strands combined with the cross-linking core;
  • the cross-linking unit II is prepared, and the cross-linking unit II is formed by the sequence complementation of the long-chain single-stranded nucleic acid chain and the complementary chain; the cross-linking unit II includes at least 2 repeating fragments, and the repeating fragments include the single-stranded first A triple nucleic acid strand and a double-stranded fourth nucleic acid strand, the cross-linking unit II is formed by alternately connecting the third nucleic acid strand and the fourth nucleic acid strand;
  • the end of the first nucleic acid strand away from the scaffold core is a sticky end, and is complementary to the sequence of the second nucleic acid strand away from the sticky end of the cross-linking core; the third nucleic acid strand is the same as the second nucleic acid strand.
  • the sequences of the cohesive ends of a nucleic acid strand are complementary.
  • the scaffold units are respectively cross-linked with the cross-linking unit I and the cross-linking unit II to obtain a nucleic acid hydrogel.
  • the method for preparing a nucleic acid hydrogel according to the present disclosure wherein the long-chain single-stranded nucleic acid chain is alternately connected by the single-stranded region N1 and the single-stranded region N2, and the long-chain single-stranded nucleic acid chain is alternately connected by the single-stranded region N1 and the single-stranded region N2
  • the nucleic acid strand includes at least 2 single-stranded regions N1 and at least 2 single-stranded regions N2;
  • the long-chain single-stranded nucleic acid strand and the complementary strand are mixed in a gel matrix and subjected to annealing treatment, and the complementary strand is complementary to the sequence of the single-stranded region N1 to form a double-stranded fourth nucleic acid strand.
  • Region N2 forms the third nucleic acid chain, and obtains the cross-linking unit II in which the fourth nucleic acid chain and the third nucleic acid chain are alternately connected;
  • a long-chain single-stranded nucleic acid chain is prepared;
  • long single-stranded nucleic acid strands are prepared in the presence of pyrophosphatase.
  • the method for preparing a nucleic acid hydrogel according to the present disclosure wherein the cross-linking unit is based on the total number of moles of repeating fragments in the cross-linking unit I and the cross-linking unit II.
  • the content of repeating fragments in II is 0.5-99%; optionally, the content of repeating fragments in the cross-linking unit II is 1-20%; preferably, the content of repeating fragments in the cross-linking unit II is 2.5-20% 10%.
  • the method for preparing a nucleic acid hydrogel according to the present disclosure wherein the materials forming the scaffold core or the cross-linked core are independently selected from the group consisting of nucleic acids, polypeptides, Polymer compounds and nanoparticles.
  • the method for preparing a nucleic acid hydrogel according to the present disclosure wherein the nucleic acid strand of any one of the first nucleic acid strand, the second nucleic acid strand, the third nucleic acid strand and the fourth nucleic acid strand any nucleotide is a modified nucleotide or an unmodified nucleotide;
  • any nucleotide of any one of the first nucleic acid strand, the second nucleic acid strand, the third nucleic acid strand and the fourth nucleic acid strand is a deoxyribonucleotide or a ribonucleotide.
  • the length of the sticky end of any one of the first nucleic acid strand and the second nucleic acid strand is 4 nt or more, preferably 4 nt -30nt, more preferably 4-20nt;
  • the length of the third nucleic acid strand is 4 nt or more, preferably 4-30 nt, more preferably 4-20 nt.
  • the length of the fourth nucleic acid strand is 4 nt or more, preferably 10-40 nt, more preferably 20-30 nt.
  • the present disclosure also provides the use of the nucleic acid hydrogel according to the present disclosure, or the nucleic acid hydrogel prepared by the method according to the present disclosure, in at least one of the following (a)-(c):
  • the nucleic acid hydrogel provided by the present disclosure is formed by cross-linking the scaffold unit with the cross-linking unit I and the cross-linking unit II, respectively, and has a spatial structure of a three-dimensional network.
  • the cross-linking unit II and the cross-linking unit I The synergistic construction with the scaffold unit realizes the effective control of the mechanical properties of the nucleic acid hydrogel and improves the mechanical strength and stability of the nucleic acid hydrogel.
  • nucleic acid hydrogels maintain the dynamic properties of supramolecular hydrogels, with rapid prototyping, shear thinning, injectability, self-healing, thermoplastic properties, and good biocompatibility. It has important application prospects in biomedical fields such as drug delivery, cell culture differentiation, protein production, and immune regulation, as well as flexible electronics such as wearable devices, artificial skin, and soft robots.
  • the nucleotides that make up the first nucleic acid strand, the second nucleic acid strand, the third nucleic acid strand, or the fourth nucleic acid strand can be modified nucleotides by introducing into the nucleus
  • the modification of nucleotides can further improve the stability of nucleic acid hydrogels, improve its immunogenicity, and broaden the application scope of nucleic acid hydrogels in the field of biomedicine.
  • the present disclosure provides a method for preparing nucleic acid hydrogels, which has the advantages of simple preparation steps and enabling rapid prototyping of nucleic acid hydrogels.
  • Figure 1 shows a schematic diagram of the process of preparing nucleic acid hydrogels
  • Figure 1A shows a DNA hydrogel prepared with scaffold units, cross-linking unit I
  • Figure 1B shows a scaffold unit, cross-linking unit I, cross-linking The mechanically enhanced DNA hydrogel prepared by unit II;
  • Figure 2 shows the rheological property test results of the nucleic acid hydrogels prepared in the various embodiments and comparative examples of the present disclosure
  • Fig. 3 shows the comparison of the mechanical properties of the nucleic acid hydrogels prepared in Example 2 of the present disclosure before and after self-healing;
  • Figure 4 shows the injectability of the nucleic acid hydrogel prepared in Example 2 of the present disclosure.
  • numerical range represented by "numerical value A to numerical value B" refers to the range including the numerical values A and B at the endpoints.
  • multiple in “multiple”, “plurality”, “plurality”, etc. means a numerical value of 2 or more.
  • the "substantially”, “substantially” or “substantially” means that the error is less than 5%, or less than 3% or less than 1% compared to the relevant perfect standard or theoretical standard.
  • water includes tap water, deionized water, distilled water, double-distilled water, purified water, ion-exchanged water, and the like, any practicable water.
  • modification of nucleotides includes one or more of modification of ribose, modification of base, and modification of phosphodiester bond.
  • the nucleotide modifications can be LNA, 2'-OMe, 3'-OMeU, vmoe, Phosphorothioate (PS), m6A , ⁇ , m1A , and the like.
  • polypeptide polypeptide
  • peptide protein
  • polymers of amino acids of any length can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids.
  • the term also includes amino acid polymers that have been modified (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).
  • a first aspect of the present disclosure provides a nucleic acid hydrogel, wherein the nucleic acid hydrogel includes a scaffold unit, and a cross-linking unit I and a cross-linking unit II respectively cross-linked with the scaffold unit;
  • the scaffold unit includes a scaffold core and at least 3 first nucleic acid strands combined with the scaffold core, and the cross-linking unit 1 includes a cross-linked core and at least 2 second nucleic acid strands combined with the cross-linked core;
  • One end of the first nucleic acid strand away from the scaffold core is a sticky end, and is complementary to the sequence of the second nucleic acid strand away from the sticky end of the cross-linked core;
  • the cross-linking unit II includes at least two repeating segments, the repeating segments include a single-stranded third nucleic acid strand and a double-stranded fourth nucleic acid strand, and the cross-linking unit II is composed of the third nucleic acid strand and the double-stranded fourth nucleic acid strand.
  • the fourth nucleic acid strand is alternately connected, and the third nucleic acid strand is complementary to the sequence of the cohesive end of the first nucleic acid strand.
  • FIG. 1A shows a Y-shaped DNA monomer (Y-DNA, also known as the scaffold unit) and the linker DNA (Linker, that is, cross-linking unit I) cross-linked DNA hydrogel.
  • Y-DNA also known as the scaffold unit
  • Linker that is, cross-linking unit I
  • the Y-DNA has three short arms connected to each other, and the one near the connecting position in the short arms is dsDNA, and the free end of dsDNA has a piece of ssDNA, which is a sticky end.
  • the 5' and 3' ends of the adaptor DNA are respectively designed with cohesive ends complementary to the ssDNA sequence of the Y-DNA monomer.
  • the Y-DNA monomer and the adaptor DNA can be cross-linked by complementary base pairing to form a three-dimensional network structure. DNA hydrogels.
  • DNA hydrogel obtained by this molecular design improves the controllability of the mechanical properties of the nucleic acid hydrogel to a certain extent.
  • the Y-DNA monomer and the connecting DNA realize self-assembly through non-covalent bonds, and the stability of the cross-linking point is insufficient, and the stability and mechanical strength of the DNA hydrogel need to be further improved.
  • the cohesive end of the first nucleic acid strand in the scaffold unit is complementary to the sequence of the cohesive end of the second nucleic acid strand in the cross-linking unit I, so that the scaffold unit and the cross-linking unit I can be cross-linked to Construction of hydrogels with three-dimensional network structure.
  • the cohesive end of the third nucleic acid strand in the cross-linking unit II is complementary to the sequence of the cohesive end of the first nucleic acid strand in the scaffold unit, realizing the mutual cross-linking of the cross-linking unit II and the scaffold unit, and the three jointly participate in the construction of nucleic acid water.
  • Three-dimensional structure of the gel (Fig. 1B).
  • the cross-linking unit II is introduced into the hydrogel structure, it is equivalent to connecting the scaffold unit and the hydrogel structure formed by cross-linking of the cross-linking unit I in the form of covalent bonds, thereby synergistically improving the cross-linking of nucleic acid hydrogels.
  • the stability of the junction can better inhibit the sliding between nucleic acid strands, and can compensate for the instability caused by the gap of the complementary sticky ends, so that the nucleic acid hydrogel has higher mechanical strength and stability.
  • the present disclosure found that the nucleic acid hydrogel formed by the joint cross-linking of the scaffold unit, the cross-linking unit I, and the cross-linking unit II not only has the full rigid network structure of the nucleic acid hydrogel, but also retains supramolecular water.
  • the dynamic properties of the gel for example, can have the dynamic properties of supramolecular hydrogels, for example, with good shear thinning, with injectable, fast self-healing, stimulus-responsive properties.
  • nucleic acid hydrogel in the present disclosure does not need to add guest molecules to construct a double network hydrogel in the process of improving its mechanical properties and maintaining the dynamic properties of the supramolecular hydrogel.
  • the nucleic acid hydrogel in the present disclosure has the advantages of easy Preparation, fast response characteristics.
  • the scaffold unit in the present disclosure includes a scaffold core and at least 3 first nucleic acid strands bound to the scaffold core.
  • the number of the first nucleic acid strands can be 3, 4, 5 and so on.
  • the first nucleic acid strand is bound to the scaffold core so that the scaffold unit forms a nucleic acid monomer structure around which 3, 4, 5, etc. short nucleic acid arms are attached.
  • the number of the first nucleic acid strands is 3, and the 3 first nucleic acid strands are combined on the core of the scaffold to obtain a Y-shaped nucleic acid monomer structure, which is conducive to the construction of a nucleic acid hydrogel with high spatial structural stability glue.
  • the material forming the scaffold core can be nucleic acids, polypeptides, polymer compounds or nanoparticles.
  • the scaffold core can also be formed from other types of biomaterials in the art, as long as they are suitable for use in the construction of nucleic acid hydrogels.
  • the scaffold core is a nucleic acid.
  • Nucleic acids serving as scaffold cores in the present disclosure may be formed by the polymerization of one or both of deoxyribonucleotides and ribonucleotides.
  • any nucleotides constituting the nucleic acid may be modified or unmodified nucleotides.
  • nucleic acids can assume different spatial conformations.
  • the nucleic acid can be an L-nucleic acid or a D-nucleic acid.
  • the scaffold core is DNA formed from deoxyribonucleotides.
  • the DNA serving as the core of the scaffold has complementary paired scaffold double-stranded regions, and the number of the scaffold double-stranded regions is set corresponding to the number of the first nucleic acid strands.
  • the two ends of each scaffold double-stranded region are respectively connected to other scaffold double-stranded regions and the first nucleic acid strand.
  • one end of the scaffold double-stranded region connected to other scaffold double-stranded regions is called a connecting end.
  • the double-stranded regions of the scaffolds are connected to each other through the connecting ends to form a scaffold core with a Y-shaped, X-shaped structure, etc., and each scaffold double-stranded region is connected to a first nucleic acid strand at one end away from the connecting ends.
  • the length of the double-stranded region of the scaffold is 4 bp or more, preferably 4-150 bp, preferably 5-50 bp, more preferably 6-30 bp, more preferably 8-20 bp.
  • the scaffold core is a polypeptide
  • the polypeptide is formed by connecting two or more amino acids with each other by peptide bonds, which can cover dipeptides, tripeptides, tetrapeptides, oligopeptides, proteins, and the like.
  • the scaffold cores are nanoparticles, which are microscopic particles on the nanometer scale, generally referring to particles smaller than 100 nm in at least one dimension.
  • semiconductor nanoparticles smaller than 10 nm are also called quantum dots due to the quantization of their electron energy levels.
  • Nanoparticles as the core of the scaffold include quantum dots, Fe 2 O 3 , Si, SiO 2 , Au, and Ag nanoparticles, among others.
  • the scaffold core is a macromolecular compound, which is a compound having a relative molecular mass of several thousand to several million by linking simple building blocks in a repeating manner.
  • the high molecular compound as the core of the scaffold covers polylactic acid, polylactic acid-co-glycolic acid copolymer, polyethylene glycol, and the like.
  • first nucleic acid strand For the first nucleic acid strand, one end of the first nucleic acid strand is connected to the scaffold core, and one end away from the scaffold core is a sticky end.
  • the cohesive end of the first nucleic acid strand is complementary to the sequence of the cohesive end of the second nucleic acid strand and is complementary to the sequence of the third nucleic acid strand.
  • the sticky end of the first nucleic acid strand is paired with base complementarity with the sticky end of the second nucleic acid strand, and the sticky end of the first nucleic acid strand is The end can be paired with the bases of the third nucleic acid strand complementary, so that the scaffold unit, the cross-linking unit I and the cross-linking unit II self-assemble to obtain a nucleic acid hydrogel.
  • the first nucleic acid strand is a single-stranded nucleic acid strand (ssDNA), and the entire first nucleic acid strand serves as a sticky end to achieve mutual cross-linking with the cross-linking unit I and the cross-linking unit II.
  • the first nucleic acid strand can also be a double-stranded nucleic acid strand, and the end of the double-stranded first nucleic acid strand away from the scaffold core extends out of ssDNA as a sticky end.
  • any one of the first nucleic acid strands has the same nucleic acid sequence, and its cohesive ends are complementary to the cohesive ends of the second nucleic acid strand in the cross-linking unit I, and the sequences of the third nucleic acid strand.
  • the sticky ends of any one of the first nucleic acid strands have the same nucleic acid sequence and are complementary to the sequences of the sticky ends of the second nucleic acid strand and the third nucleic acid strand in the cross-linking unit I.
  • the sequence of the first nucleic acid strand, or the sequence of the sticky end of the first nucleic acid strand can also have two or more choices, as long as the sticky end of the second nucleic acid strand in the corresponding cross-linking unit I
  • the sequence and the sequence of the third nucleic acid strand can be complementary to it.
  • the length of the sticky end of the first nucleic acid strand is 4 nt or more, which is beneficial for it to be in a stable cross-linked state under physiological conditions.
  • the length of the sticky end of the first nucleic acid strand is 150 nt or less, preferably 50 nt or less, more preferably 30 nt or less, and more preferably 20 nt or less.
  • the length of the sticky end of the first nucleic acid strand is preferably 4-30 nt, more preferably 4-20 nt.
  • the length of the sticky end of the first nucleic acid strand will also affect the mechanical strength of the nucleic acid hydrogel obtained by the final cross-linking. Improving the mechanical strength of nucleic acid hydrogels in the case of self-assembly.
  • the cross-linking unit I in the present disclosure includes a cross-linking core and at least 2 second nucleic acid strands bound to the cross-linking core.
  • the number of the first nucleic acid strands can be 2, 3, 4 and so on.
  • the second nucleic acid strand is bound to the cross-linking core, causing the cross-linking unit I to form a nucleic acid adaptor structure around which 2, 3, 4, etc. nucleic acid short arms are attached.
  • the number of the first nucleic acid strands is two, and the two first nucleic acid strands are bound to the cross-linked core to form a nucleic acid adaptor structure, which is conducive to constructing a nucleic acid hydrogel with high spatial structural stability.
  • the material forming the cross-linking core can be nucleic acid, polypeptide, polymer compound or nanoparticle.
  • the cross-linked core can also be formed from other types of biomaterials in the art, as long as they are suitable for use in the construction of nucleic acid hydrogels.
  • the cross-linked core is a nucleic acid.
  • Nucleic acids serving as cross-linking cores in the present disclosure may be formed by the polymerization of one or both of deoxyribonucleotides and ribonucleotides. Furthermore, any nucleotides constituting the nucleic acid may be modified or unmodified nucleotides.
  • nucleic acids can assume different spatial conformations.
  • the nucleic acid can be an L-nucleic acid or a D-nucleic acid.
  • the cross-linked core is DNA formed from deoxyribonucleotides.
  • the DNA serving as the cross-linking core has complementary paired cross-linked double-stranded regions, and both ends or one end of the cross-linked double-stranded region are connected with the second nucleic acid strand to form a cross-linked unit I.
  • the number of cross-linked double-stranded regions can be 1, 2, 3, etc.
  • the number of the cross-linked double-stranded region is 1, and the two ends thereof are respectively connected to a second nucleic acid strand to form a cross-linked unit I.
  • the length of the cross-linked double-stranded region is 4 bp or more, preferably 4-150 bp, preferably 5-50 bp, more preferably 6-30 bp, more preferably 8-20 bp.
  • the cross-linking core is a polypeptide
  • the polypeptide is formed by connecting two or more amino acids to each other by peptide bonds, which can cover dipeptides, tripeptides, tetrapeptides, oligopeptides, proteins, and the like.
  • the crosslinked cores are nanoparticles, which are microscopic particles on the nanometer scale, generally referring to particles smaller than 100 nm in at least one dimension.
  • semiconductor nanoparticles smaller than 10 nm are also called quantum dots due to the quantization of their electron energy levels.
  • Nanoparticles as crosslinking cores include quantum dots, Fe2O3 , Si, SiO2 , Au, and Ag nanoparticles, among others.
  • the cross-linking core is a polymer compound, which is a compound having a relative molecular mass of several thousand to several million by linking simple structural units in a repeating manner.
  • the high molecular compound as the crosslinking core encompasses polylactic acid, polylactic acid-glycolic acid copolymer, polyethylene glycol, and the like.
  • one end of the second nucleic acid strand is connected to the cross-linked core, and the end of the second nucleic acid strand away from the cross-linked core is a sticky end.
  • the cohesive end of the second nucleic acid strand is complementary to the sequence of the cohesive end of the first nucleic acid strand, and the mutual cross-linking between the cross-linking unit I and the scaffold unit is realized by means of base complementary pairing.
  • the second nucleic acid strand is a single-stranded nucleic acid strand (ssDNA), and the whole of the second nucleic acid strand serves as a sticky end to achieve mutual cross-linking with the scaffold unit.
  • the second nucleic acid strand can also be a double-stranded nucleic acid strand, and the end of the double-stranded second nucleic acid strand away from the cross-linked core extends out of ssDNA as a sticky end.
  • either second nucleic acid strand has the same nucleic acid sequence and its cohesive ends are complementary to the sequence of the cohesive ends of the first nucleic acid strand in the scaffold unit.
  • the sticky ends of any second nucleic acid strand have the same nucleic acid sequence and are complementary to the sequence of the sticky ends of the first nucleic acid strand in the scaffold unit.
  • the sequence of the second nucleic acid strand or the sequence of the sticky end of the second nucleic acid strand can also have two or more choices, as long as the sticky end sequence of the first nucleic acid strand in the corresponding scaffold unit can It can be achieved to complement it.
  • the length of the sticky end of the second nucleic acid strand is 4 nt or more, which is beneficial for it to be in a stable cross-linked state under physiological conditions.
  • the length of the sticky end of the second nucleic acid strand is 150 nt or less, preferably 50 nt or less, more preferably 30 nt or less, and more preferably 20 nt or less.
  • the length of the sticky end of the second nucleic acid strand is preferably 4-30 nt, more preferably 4-20 nt.
  • the length of the sticky end of the second nucleic acid strand will also affect the mechanical strength of the nucleic acid hydrogel obtained by final cross-linking. By designing the length of the sticky end in the range of 4-20nt, the nucleic acid hydrogel can be realized Improving the mechanical strength of nucleic acid hydrogels in the case of self-assembly.
  • the cross-linking unit II includes at least 2 repeating segments, the repeating segments include a single-stranded third nucleic acid strand and a double-stranded fourth nucleic acid strand, and the cross-linking unit II is composed of the third nucleic acid strand and the fourth nucleic acid strand.
  • the nucleic acid strands are alternately linked, and the third nucleic acid strand is complementary to the sequence of the cohesive end of the first nucleic acid strand.
  • each repeating fragment includes a single-stranded third nucleic acid strand and a double-stranded fourth nucleic acid strand, and the third nucleic acid strand and the fourth nucleic acid strand are alternately connected to obtain at least two third nucleic acid strands and A cross-linking unit II formed by alternately connecting at least two fourth nucleic acid strands.
  • the number of the third nucleic acid strands can be at least 2, at least 10, at least 50, at least 100, at least 200, at least 300, at least 500, at least 800, at least 1000, at least 1200, at least 1500, etc.
  • the number of the fourth nucleic acid strand can be at least 2, at least 10, at least 50, at least 100, at least 200, at least 300, at least 500, at least 800, At least 1000, at least 1200, at least 1500, etc.
  • the present disclosure does not enumerate the number of the third nucleic acid strand and the number of the fourth nucleic acid strand, as long as it is suitable for connecting the scaffold unit and the nucleic acid hydrogel structure of the cross-linking unit I synergistically, so as to make the nucleic acid hydrogel
  • the amount of glue that has both improved mechanical properties and maintains the dynamic properties of supramolecular hydrogels is sufficient.
  • the number of the third nucleic acid strand is at least 100, and the number of the fourth nucleic acid strand is at least 100.
  • the number of cross-linking sites provided by the cross-linking unit II can be adjusted, thereby realizing the network structure and structure of the nucleic acid hydrogel. Stability adjustment.
  • the third nucleic acid chain is a single-stranded nucleic acid chain
  • the fourth nucleic acid chain is a double-stranded nucleic acid chain, that is, the cross-linking unit II is formed by alternately connecting the double-stranded region and the single-stranded region. long-chain cross-linking units.
  • the sequence of the third nucleic acid strand is complementary to the sequence of the sticky end of the first nucleic acid strand in the scaffold unit, providing a cross-linking site between the cross-linking unit II and the scaffold unit.
  • the scaffold units be synergistically connected, but also the self-assembly within the sequence in the cross-linking unit II can be avoided, so as to further improve the structural stability of the nucleic acid hydrogel and effectively inhibit the interstrand of nucleic acid. Sliding to obtain a nucleic acid hydrogel with both mechanical strength and supramolecular hydrogel dynamic properties.
  • the cross-linking unit II is an extended long nucleic acid chain.
  • the ductile structure of the cross-linking unit II is more suitable for cross-linking with the scaffold single-strand, realizing the synergistic connection of the hydrogel structure formed by the cross-linking of the scaffold unit and the cross-linking unit I, and better inhibiting the sliding between nucleic acid strands. Compensates for instability caused by gaps in complementary cohesive ends.
  • the formation of the third nucleic acid strand or the fourth nucleic acid strand may be formed by the polymerization of one or both of deoxyribonucleotides and ribonucleotides, respectively. Furthermore, any nucleotides constituting the third nucleic acid strand or the fourth nucleic acid strand may be modified or unmodified nucleotides.
  • the nucleic acid structure of the third nucleic acid strand or the fourth nucleic acid strand may exhibit different spatial conformations.
  • the nucleic acid can be an L-nucleic acid or a D-nucleic acid.
  • the third nucleic acid strand is single-stranded DNA formed from deoxyribonucleotides; in some embodiments, the fourth nucleic acid strand is double-stranded DNA formed from deoxyribonucleotides.
  • the length of the third nucleic acid strand is 4 nt or more, preferably, the length of the third nucleic acid strand is 150 nt or less, preferably 50 nt or less, more preferably 30 nt or less, more preferably 20 nt or less. Specifically, the length of the third nucleic acid strand is preferably 4-30 nt, more preferably 4-20 nt. In some embodiments, the length of the fourth nucleic acid strand is 4 nt or more, preferably 10-40 nt, more preferably 20-30 nt.
  • the content of the repeating fragments is 0.5-99%;
  • the content is 1-20%; preferably, the content of the repeating segment is 2.5-10%.
  • the content of the repeating fragment is 2.5%, 5%, 7.5%, 10%, 12%, 15%, 17%, 20% and the like.
  • the second aspect of the present disclosure provides the method for preparing a nucleic acid hydrogel according to the first aspect of the present disclosure, including the step of cross-linking the scaffold unit with the cross-linking unit I and the cross-linking unit II.
  • the preparation method of nucleic acid hydrogel comprises the steps:
  • cross-linking unit I and cross-linking unit II in a gel matrix to obtain a cross-linking unit solution
  • the scaffold unit solution is mixed with the cross-linking unit solution, and the scaffold unit is self-assembled with the cross-linking unit I and the cross-linking unit II to obtain a nucleic acid hydrogel.
  • the scaffold core in the scaffold unit is nucleic acid
  • the scaffold unit can be prepared by complementary pairing of nucleic acid fragments.
  • the scaffold unit is obtained by using nucleic acid as the scaffold core and combining three first nucleic acid strands on the scaffold core.
  • three short-chain nucleic acid fragments are involved and named Y1, Y2 and Y3.
  • the nucleic acid fragment Y1 includes the single-stranded region Y11, the single-stranded region Y12 and the single-stranded region Y13 connected in sequence
  • the nucleic acid fragment Y2 includes the single-stranded region Y21, the single-stranded region Y22 and the single-stranded region Y23 connected in sequence
  • the nucleic acid fragment Y3 includes Single-chain region Y31, single-chain region Y32, and single-chain region Y33 connected in sequence.
  • sequence of the single-stranded region Y12 and the sequence of the single-stranded region Y33 are complementary sequences
  • sequence of the single-stranded region Y13 and the sequence of the single-stranded region Y22 are complementary sequences
  • sequence of the single-stranded region Y23 and the sequence of the single-stranded region Y32 are complementary sequence.
  • the nucleic acid fragment Y1, the nucleic acid fragment Y2 and the nucleic acid fragment Y3 are placed in the same reaction system, and after annealing treatment, a scaffold unit with three first nucleic acid strands can be obtained.
  • the complementary pairing of the single-stranded region Y12 of the nucleic acid fragment Y1 and the single-stranded region Y33 of the nucleic acid fragment Y3 is the double-stranded region of the scaffold
  • the complementary pairing of the single-stranded region Y13 of the nucleic acid fragment Y1 and the single-stranded region Y22 of the nucleic acid fragment Y2 is In the double-stranded region of the scaffold, the single-stranded region Y23 of the nucleic acid fragment Y2 is complementary to the single-stranded region Y32 of the nucleic acid fragment Y3 to form the double-stranded region of the scaffold, which together constitute the scaffold core of the scaffold unit.
  • the aqueous medium can be water, or a water-based buffer.
  • the gel matrix may also be an aqueous medium in any other condition capable of forming a nucleic acid hydrogel, which is not specifically limited in the present disclosure.
  • the temperature is lowered until the nucleic acid fragment Y1, the nucleic acid fragment Y2 and the nucleic acid fragment Y3 are complementary and paired to form a scaffold unit with a double-stranded region.
  • the incubation temperature, incubation time, and cooling rate are determined by the specific sequence structures of nucleic acid fragment Y1, nucleic acid fragment Y2 and nucleic acid fragment Y3, as long as the three can form a scaffold unit by hybridization and pairing.
  • the scaffold core is a polypeptide
  • the scaffold unit is prepared by covalently binding the first nucleic acid strand to the polypeptide serving as the scaffold core.
  • the scaffold unit is prepared by a click reaction, more preferably a copper catalyzed click reaction.
  • the scaffold unit is prepared by covalently binding the first nucleic acid strand to the nanoparticle serving as the scaffold core.
  • the cross-linking core in the cross-linking unit I is a nucleic acid
  • the preparation of the cross-linking unit can be obtained by complementary pairing of nucleic acid fragments.
  • the cross-linking unit is obtained by using nucleic acid as the cross-linking core, and combining two nucleic acid chains on the cross-linking core. Corresponding to this structure, two short-chain nucleic acid fragments are involved and named L1 and L2.
  • the nucleic acid fragment L1 includes the single-stranded region L11 and the single-stranded region L12 connected in sequence
  • the nucleic acid fragment L2 includes the single-stranded region L21 and the single-stranded region L22 connected in sequence.
  • sequence of the single-stranded region L12 and the sequence of the single-stranded region L22 are complementary sequences
  • the sequence of the single-stranded region L11 is a complementary sequence to at least one of the single-stranded region Y11, the single-stranded region Y21 and the single-stranded region Y31 in the scaffold unit
  • the sequence of the single-stranded region L12 is complementary to at least one of the single-stranded region Y11, the single-stranded region Y21 and the single-stranded region Y31 in the scaffold unit.
  • the mutual cross-linking of the scaffold unit and the cross-linking unit I can be achieved by complementary pairing of the sequences of the single-stranded region L11, the single-stranded region L12 and the single-stranded region Y11, the single-stranded region Y21 and the single-stranded region Y31.
  • the sequence of the single-stranded region L11 is the same as the sequence of the single-stranded region L12
  • the sequences of the single-stranded region Y11, the single-stranded region Y21 and the single-stranded region Y31 are the same
  • the sequences of the single-stranded regions L11 and L12 are the same as those of the single-stranded region L11 and L12.
  • the sequences of chain regions Y11, Y12, and Y13 are complementary to each other.
  • the nucleic acid fragment L1 and the nucleic acid fragment L2 are placed in the same reaction system, and after annealing treatment, a cross-linked unit I with two second nucleic acid strands can be obtained.
  • the cross-linking unit I the single-stranded region L22 of the nucleic acid fragment L1 and the single-stranded region L22 of the nucleic acid fragment L2 are complementary paired to form a double-stranded region, and the single-stranded region L11 and the single-stranded region L21 become the direction of the double-stranded region away from the connecting end.
  • the single-stranded region L11 and the single-stranded region L21 are respectively used as two second nucleic acid strands connected to the cross-linked core.
  • the single-stranded region L12 and the single-stranded region L22 are respectively provided with enzyme cleavage sites, so that the cross-linking unit I can be cleaved by the corresponding enzyme, thereby realizing the specific enzyme response of nucleic acid hydrogel .
  • the aqueous medium can be water, or a water-based buffer.
  • the gel matrix may also be an aqueous medium in any other condition capable of forming a nucleic acid hydrogel, which is not specifically limited in the present disclosure.
  • the temperature is lowered until the nucleic acid fragment L1 and the nucleic acid fragment L2 are complementary and paired to form a scaffold unit having a double-stranded region.
  • the incubation temperature, incubation time, and cooling speed are determined by the specific sequence structure of the nucleic acid fragment L1 and the nucleic acid fragment L2, as long as the two can form a cross-linking unit I through hybridization and pairing.
  • the cross-linking core is a polypeptide
  • the preparation of the cross-linking unit I is performed by covalently binding the second nucleic acid chain to the polypeptide serving as the cross-linking core.
  • the crosslinking unit is prepared by a click reaction, more preferably a copper catalyzed click reaction.
  • the preparation of the cross-linking unit is performed by covalently binding the second nucleic acid strand to the nanoparticle as the cross-linking core.
  • the step of preparing the cross-linking unit II comprises:
  • the long-chain single-stranded nucleic acid chain comprising at least 2 single-stranded regions N1 and at least 2 single-stranded regions N2;
  • the long-chain single-stranded nucleic acid strand and the complementary strand are mixed in a gel matrix and subjected to annealing treatment, and the complementary strand is complementary to the sequence of the single-stranded region N1 to form a double-stranded fourth nucleic acid strand.
  • Region N2 forms the third nucleic acid chain, and obtains the cross-linking unit II in which the fourth nucleic acid chain and the third nucleic acid chain are alternately connected; wherein, adjacent 1 single-stranded third nucleic acid chain and 1 double-stranded fourth nucleic acid chain Linked to form a repeating segment, the cross-linking unit II includes at least two repeating segments.
  • the method for realizing long-chain nucleic acid preparation in the art can be used, as long as a long-chain single-stranded nucleic acid chain formed by alternately connecting the single-stranded region N1 and the single-stranded region N2 can be obtained.
  • the long-chain single-stranded nucleic acid chain is obtained by rolling circle amplification using a circular nucleic acid chain as a template.
  • long single-stranded nucleic acid strands are obtained by chemical synthesis.
  • long single-stranded nucleic acid strands are prepared in the presence of pyrophosphatase. Specifically, inorganic pyrophosphatase is added to the reaction system of rolling circle amplification, and by adding inorganic pyrophosphatase, the by-product pyrophosphate is decomposed, and the formation of DNA/MgP 2 O 7 nanostructures is avoided.
  • the cross-linking unit II prepared with the long-chain single-stranded nucleic acid chain is further prevented from agglomerating, so that the structure of the cross-linking unit II is spread as much as possible, which is beneficial to the tandem connection of the scaffold units.
  • the linear template strand comprises 2 single-stranded regions M1 and 2 single-stranded regions M2, the single-stranded regions M1 and the single-stranded regions M2 are alternately connected to form a linear template strand, and the linear template strand is circularized processed to form a circular nucleic acid chain.
  • a circular nucleic acid chain in which the single-stranded region M1 and the single-stranded region M2 are alternately arranged is used as a template, and a rolling amplification primer is added.
  • a large number of single-stranded regions N1 and M2 are obtained.
  • the sequence of the single-stranded region N1 and the sequence of the single-stranded region M1 are complementary sequences
  • the sequence of the single-stranded region N2 and the sequence of the single-stranded region M2 are complementary sequences.
  • the sequence of the single-stranded region N2 is a complementary sequence to at least one of the single-stranded region Y11, the single-stranded region Y21 and the single-stranded region Y31 in the scaffold unit.
  • the sequence of single-stranded region N2 is the same as the sequence of single-stranded region L11 in cross-linking unit I; in some embodiments, the sequence of single-stranded region N2 is the same as the sequence of single-stranded region L21 in cross-linking unit I In some embodiments, the sequence of the single-stranded region N2 is the same as that of the single-stranded region L11 and the single-stranded region L21; and the sequences of the single-stranded region Y11, single-stranded region Y21 and single-stranded region Y31 in the scaffold unit are the same , the sequences of the single-chain regions N2, L11, and L21 are complementary to the sequences of the single-chain regions Y11, Y21, and Y31.
  • the temperature is lowered until the sequences of the complementary strand and the single-stranded region N1 are complementary paired to form a double-stranded fourth nucleic acid strand, and the single-stranded region N2 corresponds to the formation of a single-stranded fourth nucleic acid strand.
  • the incubation temperature, incubation time, and cooling rate are determined by the specific sequence structure of the single-stranded region N1 and the complementary strand, as long as the two can form a fourth nucleic acid strand by hybridization and pairing.
  • the aqueous medium can be water, or a water-based buffer.
  • the gel matrix may also be an aqueous medium in any other condition capable of forming a nucleic acid hydrogel, which is not specifically limited in the present disclosure.
  • the scaffold unit solution is mixed with the cross-linking unit solution, and the scaffold units are respectively self-assembled with the cross-linking unit I and the cross-linking unit II to obtain a nucleic acid hydrogel.
  • the scaffold unit solution is a scaffold unit solution containing scaffold units obtained by dissolving nucleic acid fragments capable of assembling to form scaffold units in a gel matrix, followed by annealing treatment.
  • the cross-linking unit solution is to dissolve long-chain single-stranded nucleic acid strands, complementary strands, and nucleic acid fragments capable of assembling to form the cross-linking unit I together in a gel matrix, and after annealing treatment, the obtained product contains Crosslinking unit solution of Crosslinking Unit I and Crosslinking Unit II.
  • the scaffold unit solution and the cross-linking unit solution are mixed at 4-50 °C, preferably 5-40 °C, more preferably 10-30 °C, so that the scaffold unit is respectively mixed with the cross-linking unit I and the cross-linking unit II.
  • Base complementary pairing enables self-assembly of nucleic acid hydrogels.
  • the scaffold unit solution is mixed with the crosslinking unit solution at pH 3-11, preferably pH 4-10, more preferably pH 5-9, still more preferably pH 6-8.
  • the molar ratio of the scaffold units in the scaffold unit solution to the total cross-link units in the cross-link unit solution is 2:1-1:3, preferably 1:1-1:2, more preferably 1:1: 1.
  • the nucleic acid hydrogel is prepared by the method in the present disclosure.
  • the nucleic acid hydrogel can be rapidly formed, has the advantages of simple preparation steps and easy realization of conditions, etc., and can realize the rapid preparation of nucleic acid hydrogel.
  • a third aspect of the present disclosure provides the use of the nucleic acid hydrogel of the first aspect, or the nucleic acid hydrogel prepared by the method of the second aspect, in at least one of the following (a)-(c):
  • nucleic acid hydrogels are suitable for use as biomedical materials in the fields of drug delivery, cell culture and differentiation, protein production, immune regulation, etc., or As a flexible electronic material, it is used in wearable devices, artificial skin, soft robots, etc., or as a three-dimensional printing material in the field of 3D printing.
  • the experimental techniques and experimental methods used in the present embodiment are conventional technical methods, such as the experimental methods that do not specify specific conditions in the following examples, usually according to conventional conditions such as people such as Sambrook, molecular cloning: experiment The conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as suggested by the manufacturer. Materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • the DNA sequences (including Y1, Y2, Y3, L1, L2, rolling circle amplification primers and complementary strands of long-chain single-stranded DNA) were prepared by standard phosphoramidite DNA solid-phase synthesis (Mermade-12 DNA synthesizer). , U.S., Bio Automation Company), and separated and purified by reversed-phase high performance liquid chromatography (Agilent 1200, U.S., Agilent Company), and the purity of the raw material was characterized by LC-MS (Shimadzu 2020, Japan).
  • the linear template chain in the embodiment is modified with a phosphate group at the 5' end, purchased from Qingke Xinye Biotechnology Co., Ltd. (Beijing, China), and purified by HPLC. All experimental water used ultrapure water produced by Millipore Company with 18.2M ⁇ cm. All other chemical reagents are of superior grade and above.
  • Step 1 using a rolling circle amplification reaction to prepare long-chain single-stranded DNA, as follows:
  • Exonuclease I and 2.5 ⁇ L of Exonuclease III were added to the above system.
  • the uncyclized linear template chain was digested by reacting at 37°C for 30min, then inactivated at 90°C for 5min, and cooled at room temperature for 10min. After the reaction product was electrophoresed on a 20% acrylamide/8M urea denaturing gel, the cyclization or ligation product was observed. Purification was carried out by ultrafiltration three times using an ultrafiltration tube with a molecular weight cut-off of 3000 Da.
  • the absorbance at 260 nm of long single-stranded DNA was measured using a UV spectrophotometer (Nanodrop 2000, Thermo Scientific). The amount of all repeats in long single-stranded DNA was calculated using the extinction coefficient at 260 nm of the repeat sequence.
  • PBS phosphate buffer
  • step 3 29.25 nmol of L1, L2, the long-chain single-stranded DNA containing 1.5 nmol repeating fragments and the complementary strand of 1.5 nmol long-chain single-stranded DNA are mixed, and the amount of the complementary chain is the same as that of the long-chain single-stranded DNA repeat sequence. The amount is equal, and the total amount of L1, L2 and long-chain single-stranded DNA repeats is 60 nmol.
  • 20 ⁇ l of phosphate buffer was added, heated at 95° C. for 5 min, and naturally cooled to room temperature to obtain a cross-linking unit solution with a molar content of repeating fragments in cross-linking unit II of 2.5%.
  • step 4 the scaffold unit solution and the cross-linking unit solution are mixed at room temperature, and after shaking and centrifuging, DNA supramolecular hydrogels with different cross-linking unit II contents, that is, nucleic acid hydrogels, are obtained.
  • Nucleic acid hydrogels were prepared by the methods shown in steps 1 to 4 in Example 1, wherein the preparation of the cross-linking unit solution in step 3 was as follows:
  • the single-stranded DNA solution containing 3.0 nmol of long-chain repeating sequences and 3.0 nmol of complementary strands are mixed and then freeze-dried to obtain a cross-linking unit II with a molar content of repeating fragments of 5.0%. unit solution.
  • Nucleic acid hydrogels were prepared by the methods shown in steps 1 to 4 in Example 1, wherein the preparation of the cross-linking unit solution in step 3 was as follows:
  • the single-stranded DNA solution containing 4.5 nmol of long-chain repeating sequences and 4.5 nmol of complementary strands are mixed and lyophilized to obtain a cross-linking unit II with a molar content of repeating fragments of 7.5%. unit solution.
  • Nucleic acid hydrogels were prepared by the methods shown in steps 1 to 4 in Example 1, wherein the preparation of the cross-linking unit solution in step 3 was as follows:
  • Nucleic acid hydrogels were prepared by the methods shown in steps 2 to 4 in Example 1, wherein the preparation of the cross-linking unit solution in step 3 was as follows:
  • Kinexus Pro+ rotational rheometer produced by Malvern, UK was used to test the rheological properties of each hydrogel sample.
  • a parallel plate rotor with a diameter of 8 mm was selected for testing, and the distance between the test table and the parallel plate was set to 150 ⁇ m. Place 40 ⁇ L of the hydrogel sample in the center of the test bench. After the rotor is pressed down, the excess gel around it is gently scraped off with a spatula, and a small amount of silicone oil is added dropwise to seal it to prevent water volatilization.
  • the storage modulus G' and the loss modulus G" of the nucleic acid hydrogels in Examples 1-4 and Comparative Example 1 were tested with a fixed temperature of 25°C, a strain amplitude of 1%, and an oscillation frequency of 1 Hz.
  • Example 1 -4 and Comparative Example 1 were tested on three parallel samples, and the statistical results are shown in Figure 2.
  • the storage modulus G' reflects the elasticity of the material, that is, the rigidity, and the greater the storage modulus, the less easily the material deforms.
  • the storage modulus G' of the nucleic acid hydrogels of each embodiment is greater than that of the comparative example hydrogels, and with the increase of the repeating fragment content in the cross-linking unit II, the nucleic acid hydrogels The G' gradually increased, indicating that the mechanical properties of nucleic acid hydrogels were effectively improved.

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Abstract

一种核酸水凝胶及其制备方法和用途。核酸水凝胶是由支架单元分别与交联单元I和交联单元II相交联形成，核酸水凝胶具有提高的力学强度与稳定性以及超分子水凝胶的动态特性。可用于药物递送、细胞培养分化、蛋白生产、免疫调控等生物医用领域，以及可穿戴设备、人造皮肤、软机器人等柔性电子领域。

Description

一种力学性能提高的核酸水凝胶及其制备方法和用途 技术领域
本公开属于高分子材料和生物技术领域，具体来说，本公开涉及一种核酸水凝胶及其制备方法和用途。
背景技术
超分子水凝胶是一种基于非共价相互作用自组装形成的具有三维网状结构的软物质材料。相比于共价交联的水凝胶而言，非共价相互作用的瞬态可逆性赋予了超分子水凝胶良好的动态特性，例如刺激响应性、可注射性、形状自适应性和自修复性能等，在生物医药、柔性电子材料以及软体机器人等众多领域展现出巨大的应用潜力。然而，超分子相互作用快速的解离和重组速率使得超分子水凝胶的力学性能较差，这就限制了超分子水凝胶作为结构材料在许多领域的应用。因此，如何设计和制备一种兼具高力学强度与动态特性的水凝胶是一个长期挑战。
目前有多种方法可以用于超分子水凝胶力学强度增强，包括引入共价交联网络、纳米复合材料掺杂等方法。其中，引入共价交联网络可以通过构建柔性和刚性的互联网络有效地耗散能量，从而保证水凝胶优异的力学性能。但由于共价交联点的不可逆性，水凝胶的动态特性将消失，另外当共价交联网络断裂时，会造成水凝胶的永久性损伤和较差的抗疲劳性能。纳米材料，例如碳纳米颗粒、量子点、金属纳米颗粒等，也可以被引入超分子水凝胶中，作为额外的交联点，在提高水凝胶力学性能的同时也可以赋予超分子水凝胶额外的物理性质。但是纳米材料的引入可能会带来未知的生物毒性。另外，这些方法在增强力学性能的同时会改变水凝胶网络的交联结构和交联密度，超分子水凝胶网络原有的拓扑结构、网孔分布和渗透性能将不再保留。
核酸分子可以编码、存储和传递遗传信息，是生命系统的核心分子之一。近年来，随着化学、材料学以及纳米技术的飞速发展，人们除了将核酸分子作为遗传信息载体进行大量的研究和探索之外，也开始将其作为一种组装材料来使用。从材料和化学的角度来看，核酸分子具有结构明确、碱基的特异性识别、易于功能化修饰等特点，在化学、生物学和材料学等领域获得了广泛的关注与发展。目前，人们利用核酸分子的优异性能，已经设计合成了多种核酸纳米材料。其中，核酸水凝胶是一种典型代表，利用核酸分子制作水凝胶，既可利用了水凝胶的骨架功能，也可利用DNA的生物功能，实现了水凝胶材料结构与功能的统一融合，在包括药物运载和缓释、生物检测、蛋白质生产和免疫调控等多个生物医学领域中具有广泛的应用。
核酸水凝胶在多领域具有广泛的应用前景，如何进一步提高核酸水凝胶的力学性能，并且在提高其力学强度的同时使核酸水凝胶具有超分子水凝胶的动态特性，是本领域当前亟需解决的重要问题。
发明内容
发明要解决的问题
鉴于现有技术中存在的问题，例如，需要在提高核酸水凝胶力学性能的同时，实现核酸水凝胶可注射、可自愈等的动态特性。为此，本公开提供了一种核酸水凝胶，由支架单元分别与交联单元I和交联单元II相交联形成，通过三者的协同配合，具有力学强度高，稳定性好的优点，在保持核酸水凝胶全刚性网络结构的同时，能够保留超分子水凝胶的动态特性，在生物医药、柔性电子等领域均有重要应用前景。
用于解决问题的方案
本公开首先提供了一种核酸水凝胶，其中，所述核苷酸水凝胶包括支架单元，以及分别与所述支架单元相交联的交联单元I和交联单元II；
所述支架单元包括支架核心和与所述支架核心结合的至少3个第一核酸链，所述交联单元I包括交联核心和与所述交联核心结合的至少2个第二核酸链；所述第一核酸链远离所述支架核心的一端为粘性末端，且与所述第二核酸链远离所述交联核心的粘性末端的序列互补；
所述交联单元II包括至少2个重复片段，所述重复片段包括单链的第三核酸链和双链的第四核酸链，所述交联单元II由所述第三核酸链和所述第四核酸链交替连接形成，且所述第三核酸链与所述第一核酸链的粘性末端的序列互补。
在一些实施方式中，根据本公开所述的核酸水凝胶，其中，以所述交联单元I和所述交联单元II中重复片段的总摩尔数计，所述重复片段的含量为0.5-99％；可选地，所述重复片段的含量为1-20％；优选地，所述重复片段的含量为2.5-10％。
在一些实施方式中，根据本公开所述的核酸水凝胶，其中，形成所述支架核心或所述交联核心的材料彼此独立地选自由如下材料组成的组：核酸、多肽、高分子化合物和纳米颗粒。
在一些实施方式中，根据本公开所述的核酸水凝胶，其中，所述第一核酸链、第二核酸链、第三核酸链和第四核酸链中的任一核酸链的任一核苷酸为修饰的核苷酸或未修饰的核苷酸；
可选地，所述第一核酸链、第二核酸链、第三核酸链和第四核酸链中的任一核酸链的任一核苷酸为脱氧核糖核苷酸或核糖核苷酸。
在一些实施方式中，根据本公开所述的核酸水凝胶，其中，所述第一核酸链、第二核酸链中的任一核酸链的粘性末端的长度为4nt以上，优选4-30nt，更优选4-20nt；
可选地，所述第三核酸链的长度为4nt以上，优选4-30nt，更优选4-20nt。
在一些实施方式中，根据本公开所述的核酸水凝胶，其中，所述第四核酸链的长度为4nt以上，优选10-40nt，更优选为20-30nt。
本公开还提供了一种制备根据本公开所述的核酸水凝胶的方法，其中，所述方法包括使支架单元与交联单元I和交联单元II交联成型的步骤。
在一些实施方式中，根据本公开所述的方法，其中，所述方法包括如下步骤：
在凝胶基质中制备支架单元，得到支架单元溶液；
在凝胶基质中制备交联单元I和交联单元II，得到交联单元溶液；
将所述支架单元溶液与所述交联单元溶液混合，所述支架单元与交联单元I和交联单元II自组装，得到核酸水凝胶。
在一些实施方式中，根据本公开所述的方法，其中，制备所述交联单元II的步骤包括：
制备由单链区N1和单链区N2交替连接的长链的单链核酸链，所述长链的单链核酸链包括至少2个单链区N1和至少2个单链区N2；
所述长链的单链核酸链与互补链在凝胶基质中混合，退火处理，所述互补链与所述单链区N1的序列互补，形成双链的第四核酸链，所述单链区N2形成第三核酸链，得到第四核酸链和第三核酸链交替连接的交联单元II；
可选地，以环状的核酸链为模板，通过滚环扩增，制备长链的单链核酸链；
优选地，在焦磷酸酶存在的环境下，制备长链的单链核酸链。
本公开还提供了一种制备核酸水凝胶的方法，其中，包括以下步骤：
制备支架单元，所述支架单元包括支架核心和与所述支架核心结合的至少3个第一核酸链；
制备交联单元I，所述交联单元I包括交联核心和与所述交联核心结合的至少2个第二核酸链；
制备交联单元II，所述交联单元II由长链的单链核酸链与互补链的序列互补形成；所述交联单元II包括至少2个重复片段，所述重复片段包括单链的第三核酸链和双链的第四核酸链，所述交联单元II由所述第三核酸链和所述第四核酸链交替连接形成；
其中，所述第一核酸链远离所述支架核心的一端为粘性末端，且与所述第二核酸链远离所述交联核心的粘性末端的序列互补；所述第三核酸链与所述第一核酸链的粘性末端的序列互补。
在凝胶基质中，所述支架单元分别与所述交联单元I和所述交联单元II相交联，得到核酸水凝胶。
在一些实施方式中，根据本公开所述的制备核酸水凝胶的方法，其中，长链的单链核酸链由单链区N1和单链区N2交替连接的，所述长链的单链核酸链包括至少2个单链区N1和至少2个单链区N2；
所述长链的单链核酸链与互补链在凝胶基质中混合，退火处理，所述互补链与所述单链区N1的序列互补，形成双链的第四核酸链，所述单链区N2形成第三核酸链，得到第四核酸链和第三核酸链交替连接的交联单元II；
可选地，以环状的核酸链为模板，通过滚环扩增，制备长链的单链核酸链；
优选地，在焦磷酸酶存在的环境下，制备长链的单链核酸链。
在一些实施方式中，根据本公开所述的制备核酸水凝胶的方法，其中，以所述交联单元I和所述交联单元II中重复片段的总摩尔数计，所述交联单元II中重复片段的含量为0.5-99％；可选地，所述交联单元II中重复片段的含量为1-20％；优选地，所述交联单元II中重复片段的含量为2.5-10％。
在一些实施方式中，根据本公开所述的制备核酸水凝胶的方法，其中，形成所述支架核心或所述交联核心的材料彼此独立地选自由如下材料组成的组：核酸、多肽、高分子化合物和纳米颗粒。
在一些实施方式中，根据本公开所述的制备核酸水凝胶的方法，其中，所述第一核酸链、第二核酸链、第三核酸链和第四核酸链中的任一核酸链的任一核苷酸为修饰的核苷酸或未修饰的核苷酸；
可选地，所述第一核酸链、第二核酸链、第三核酸链和第四核酸链中的任一核酸链的任一核苷酸为脱氧核糖核苷酸或核糖核苷酸。
在一些实施方式中，根据本公开所述的制备核酸水凝胶的方法，其中，所述第一核酸链、第二核酸链中的任一核酸链的粘性末端的长度为4nt以上，优选4-30nt，更优选4-20nt；
可选地，所述第三核酸链的长度为4nt以上，优选4-30nt，更优选4-20nt。
在一些实施方式中，根据本公开所述的制备核酸水凝胶的方法，其中，所述第四核酸链的长度为4nt以上，优选10-40nt，更优选为20-30nt。
本公开还提供了一种根据本公开所述的核酸水凝胶，或以根据本公开所述的方法制备的核酸水凝胶在如下(a)-(c)至少一种中的用途：
(a)作为或制备生物医用材料；
(b)作为或制备柔性电子材料；
(c)作为或制备三维打印材料。
发明的效果
在一些实施方式中，本公开提供的核酸水凝胶，是由支架单元分别与交联单元I和交联单元II相交联形成，具有三维网络的空间结构，交联单元II、交联单元I与支架单元的协同构建，实现了对核酸水凝胶的力学性能的有效控制，提高了核酸水凝胶的力学强度与稳定性。与此同时，核酸水凝胶保持有超分子水凝胶的动态特性，具有可快速成 型、剪切变稀、可注射性、自修复性、热塑性等性能，同时具有良好的生物相容性，在药物递送、细胞培养分化、蛋白生产、免疫调控等生物医用领域，以及可穿戴设备、人造皮肤、软机器人等柔性电子领域具有重要的应用前景。
在一些实施方式中，本公开提供的核酸水凝胶，组成第一核酸链、第二核酸链、第三核酸链或第四核酸链的核苷酸可以是修饰的核苷酸，通过引入核苷酸的修饰，可进一步提高核酸水凝胶的稳定性，改善其免疫原性，拓宽核酸水凝胶在生物医药领域的应用范围。
在一些实施方式中，本公开提供了核酸水凝胶的制备方法，具有制备步骤简单、能够实现核酸水凝胶快速成型的优点。
附图说明
图1示出了制备核酸水凝胶的过程示意图，图1A示出了以支架单元、交联单元I制备的DNA水凝胶，图1B示出了以支架单元、交联单元I、交联单元II制备的力学性能增强的DNA水凝胶；
图2示出了本公开各实施例和对比例中制备的核酸水凝胶的流变性能测试结果；
图3示出了本公开实施例2中制备的核酸水凝胶自愈合前后力学性质对比；
图4示出了本公开实施例2中制备的核酸水凝胶的可注射性。
具体实施方式
以下，针对本公开的内容进行详细说明。以下所记载的技术特征的说明基于本公开的代表性的实施方案、具体例子而进行，但本公开不限定于这些实施方案、具体例子。需要说明的是：
本公开中，使用“数值A～数值B”表示的数值范围是指包含端点数值A、B的范围。
本公开中，如没有特殊声明，则“多”、“多种”、“多个”等中的“多”表示2或以上的数值。
本公开中，所述“基本上”、“大体上”或“实质上”表示于相关的完美标准或理论标准相比，误差在5％以下，或3％以下或1％以下。
本公开中，如没有特别说明，则“％”均表示质量百分含量。
本公开中，使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。
本公开中，“任选的”或“任选地”是指接下来描述的事件或情况可发生或可不发生，并且该描述包括该事件发生的情况和该事件不发生的情况。
本公开中，虽然所公开的内容支持术语“或”、“或者”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，权利要求中的术语“或”、“或者”是指“和/或”。
本公开中所使用的“水”包括自来水、去离子水、蒸馏水、双蒸水、纯净水、离子交换水等任何可行的水。
在本公开中，核苷酸的修饰包括对核糖的修饰，对碱基的修饰，以及对磷酸二酯键的修饰中的一种或多种。示例性的，核苷酸的修饰可以是LNA、2’-OMe、3’-OMeU、vmoe、Phosphorothioate(PS)、m 6A、Ψ、m 1A等等。
在本公开中，术语“多肽”、“肽”和“蛋白质”在本文中互换地使用并且为任意长度的氨基酸聚合物。该聚合物可以是线形或分支的，它可以包含修饰的氨基酸，并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如，二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作，如以标记组分缀合)的氨基酸聚合物。
第一方面
本公开的第一方面提供了一种核酸水凝胶，其中，所述核酸水凝胶包括支架单元，以及分别与所述支架单元相交联的交联单元I和交联单元II；
所述支架单元包括支架核心和与所述支架核心结合的至少3个第一核酸链，所述交联单元I包括交联核心和与所述交联核心结合的至少2个第二核酸链；所述第一核酸链远离所述支架核心的一端为粘性末端，且与所述第二核酸链远离所述交联核心的粘性末端的序列互补；
所述交联单元II包括至少2个重复片段，所述重复片段包括单链的第三核酸链和双链的第四核酸链，所述交联单元II由所述第三核酸链和所述第四核酸链交替连接形成，且所述第三核酸链与所述第一核酸链的粘性末端的序列互补。
需要说明的是，在目前的核酸水凝胶中，存在以分叉状的DNA单体(branched DNA monomer，BDM)与衔接DNA交联形成的DNA水凝胶，其中，BDM可以是由3个、4个等的DNA短臂共同连接形成的X型、T型、Y型的DNA单体，图1A中示出了一种Y型DNA单体(Y-DNA，也即支架单元)与衔接DNA(Linker，也即交联单元I)交联形成的DNA水凝胶。Y-DNA具有3个相互连接的短臂，短臂中靠近连接位置的为dsDNA，dsDNA的自由端具有一段ssDNA，为粘性末端。衔接DNA的5’端和3’端分别设计有与Y-DNA单体的ssDNA序列互补的粘性末端，Y-DNA单体与衔接DNA通过碱基互补配对的方式可交联形成具有三维网络结构的DNA水凝胶。通过该分子设计得到的DNA水凝胶虽然在一定程度上提高了核酸水凝胶的力学性能的可控性。但是，Y-DNA单体与衔接DNA通过非共价键的方式实现自组装，交联点的稳定性存在不足，DNA水凝胶的稳定性及力学强度都需要进一步的提升。
本公开中的核酸水凝胶，支架单元中第一核酸链的粘性末端通过与交联单元I中第二核酸链的粘性末端的序列互补，能够使支架单元与交联单元I相交联，以构筑具有三维网络结构的水凝胶。同时，交联单元II中第三核酸链的粘性末端与支架单元中第一核酸链的粘性末端的序列互补，实现了交联单元II与支架单元的相互交联，三者共同参与构筑核酸水凝胶的三维空间结构(图1B)。而交联单元II在引入水凝胶结构后，相当于以共价键的形式将支架单元和交联单元I交联形成的水凝胶结构连接起来，从而协同的提高了核酸水凝胶交联点的稳定性，可以更好地抑制核酸链之间的滑动，并且能够弥补互补粘性末端的缺口带来的不稳定性，使核酸水凝胶具有更高的力学强度和稳定性。
与此同时，本公开发现，以支架单元、交联单元I、交联单元II共同交联形成的核酸水凝胶，不仅具有核酸水凝胶的全刚性网络结构，同时也保留了超分子水凝胶的动态特性，例如能够具有超分子水凝胶的动态特性，例如，具有良好的剪切变稀，具有可注射、快速自愈合、刺激响应性质。
此外，本公开中的核酸水凝胶，在改善其力学性能和保持超分子水凝胶动态特性的过程中，无需加入客体分子构筑双网络水凝胶，本公开中的核酸水凝胶具有易于制备、快速响应的特点。
<支架单元>
本公开中的支架单元包括支架核心和与支架核心结合的至少3个第一核酸链。示例性的，第一核酸链的个数可以是3个、4个、5个等等。第一核酸链结合于支架核心上，使支架单元形成环绕连接有3个、4个、5个等等的核酸短臂的核酸单体结构。
在一些实施方式中，第一核酸链的个数为3个，3个第一核酸链结合于支架核心上，得到Y型的核酸单体结构，有利于构筑空间结构稳定性高的核酸水凝胶。
对于支架核心，形成支架核心的材料可以是核酸、多肽、高分子化合物或纳米颗粒。此外，支架核心也可以由本领域中其他类型的生物材料形成，只要其适合用于构筑核酸水凝胶即可。
在一些实施方式中，支架核心为核酸。本公开中作为支架核心的核酸可以是由脱氧 核糖核苷酸和核糖核苷酸中的一种或两种聚合形成。并且，对于组成核酸的任意一个核苷酸，可以是修饰的或者是未修饰的核苷酸。
在本公开中，核酸可以呈现不同的空间构象。示例性的，核酸可以是L-核酸或D-核酸。
在一些具体的实施方式中，支架核心是由脱氧核糖核苷酸形成的DNA。进一步的，作为支架核心的DNA具有互补配对的支架双链区，并且支架双链区的个数对应第一核酸链的个数设置。每个支架双链区的两端分别与其它的支架双链区以及第一核酸链相连，为了便于表述，将支架双链区连接其它支架双链区的一端称为连接端。各支架双链区通过连接端互相连接形成呈Y型、X型等等结构的支架核心，并且，每个支架双链区远离连接端的一端连接一个第一核酸链。
在一些更为具体的实施方式中，支架双链区的长度为4bp以上，优选4～150bp，优选5～50bp，更优选6～30bp，更优选8～20bp。
在一些实施方式中，支架核心为多肽，多肽由两个以上的氨基酸以肽键相互连接形成，可以涵盖二肽、三肽、四肽、寡肽、蛋白质等等。
在一些实施方式中，支架核心为纳米颗粒，其为纳米量级的微观颗粒，一般指至少在一个维度上小于100nm的颗粒。具体地，小于10nm的半导体纳米颗粒由于其电子能级量子化，又被称为量子点。作为支架核心的纳米颗粒涵盖量子点、Fe 2O 3、Si、SiO 2、Au和Ag纳米颗粒等等。
在一些实施方式中，支架核心为高分子化合物，其为通过将简单的结构单元以重复的方式连接的、相对分子质量为几千到几百万的化合物。具体地，作为支架核心的高分子化合物涵盖聚乳酸、聚乳酸-羟基乙酸共聚物和聚乙二醇等等。
对于第一核酸链，第一核酸链的一端连接于支架核心上，其远离支架核心的一端为粘性末端。第一核酸链的粘性末端与第二核酸链的粘性末端的序列互补，且与第三核酸链的序列互补。当支架单元与交联单元I和交联单元II在凝胶基质中混合后，第一核酸链的粘性末端通过与第二核酸链的粘性末端的碱基互补配对，并且第一核酸链的粘性末端可以与第三核酸链的碱基互补配对，使支架单元、交联单元I和交联单元II自组装得到核酸水凝胶。
在一些实施方式中，第一核酸链为单链的核酸链(ssDNA)，第一核酸链的整体作为粘性末端，实现与交联单元I和交联单元II的相互交联。在一些实施方式中，第一核酸链也可以是双链的核酸链，并且双链的第一核酸链远离支架核心的一端延伸出ssDNA作为粘性末端。在一些实施方式中，任一第一核酸链均具有相同的核酸序列，且其粘性末端与交联单元I中第二核酸链的粘性末端，以及第三核酸链的序列互补。在一些实施方式中，任一第一核酸链的粘性末端均具有相同的核酸序列，且与交联单元I中第二核酸链的粘性末端，以及第三核酸链的序列互补。在一些可选的实施方式中，第一核酸链的序列，或者第一核酸链的粘性末端的序列也可以有两种以上的选择，只要对应的交联单元I中第二核酸链的粘性末端序列，以及第三核酸链的序列能够实现与其互补即可。通过改变粘性末端的长度或序列，可以调节核酸水凝胶的热稳定性和机械强度，从而赋予凝胶不同的温度响应性和力学性能。
在一些实施方式中，第一核酸链的粘性末端的长度为4nt以上，这样有利于其在生理条件下处于稳定交联状态。优选地，第一核酸链的粘性末端的长度为150nt以下，优选50nt以下，更优选30nt以下，更优选20nt以下。具体的，第一核酸链的粘性末端的长度优选4-30nt，更优选4-20nt。另一方面，第一核酸链的粘性末端的长度也会影响最终交联得到的核酸水凝胶的力学强度，通过设计粘性末端的长度在4-20nt的范围内，可以在实现核酸水凝胶自组装的情况下提高核酸水凝胶的力学强度。
<交联单元I>
本公开中的交联单元I包括交联核心和与交联核心结合的至少2个第二核酸链。示例性的，第一核酸链的个数可以是2个、3个、4个等等。第二核酸链结合于交联核心上，使交联单元I形成环绕连接有2个、3个、4个等等的核酸短臂的核酸衔接体结构。
在一些实施方式中，第一核酸链的个数为2个，2个第一核酸链结合于交联核心上，形成核酸衔接体结构，有利于构筑空间结构稳定性高的核酸水凝胶。
对于交联单元I，形成交联核心的材料可以是核酸、多肽、高分子化合物或纳米颗粒。此外，交联核心也可以由本领域中其他类型的生物材料形成，只要其适合用于构筑核酸水凝胶即可。
在一些实施方式中，交联核心为核酸。本公开中作为交联核心的核酸可以是由脱氧核糖核苷酸和核糖核苷酸中的一种或两种聚合形成。并且，对于组成核酸的任意一个核苷酸，可以是修饰的或者是未修饰的核苷酸。
在本公开中，核酸可以呈现不同的空间构象。示例性的，核酸可以是L-核酸或D-核酸。
在一些具体的实施方式中，交联核心是由脱氧核糖核苷酸形成的DNA。进一步的，作为交联核心的DNA具有互补配对的交联双链区，交联双链区的两端或一端与第二核酸链相连，形成交联单元I。交联双链区的个数可以是1个、2个、3个等等。示例性的，交联双链区的个数为1，其两端分别连接一个第二核酸链，形成交联单元I。
在一些更为具体的实施方式中，交联双链区的长度为4bp以上，优选4～150bp，优选5～50bp，更优选6～30bp，更优选8～20bp。
在一些实施方式中，交联核心为多肽，多肽由两个以上的氨基酸以肽键相互连接形成，可以涵盖二肽、三肽、四肽、寡肽、蛋白质等等。
在一些实施方式中，交联核心为纳米颗粒，其为纳米量级的微观颗粒，一般指至少在一个维度上小于100nm的颗粒。具体地，小于10nm的半导体纳米颗粒由于其电子能级量子化，又被称为量子点。作为交联核心的纳米颗粒涵盖量子点、Fe 2O 3、Si、SiO 2、Au和Ag纳米颗粒等等。
在一些实施方式中，交联核心为高分子化合物，其为通过将简单的结构单元以重复的方式连接的、相对分子质量为几千到几百万的化合物。具体地，作为交联核心的高分子化合物涵盖聚乳酸、聚乳酸-羟基乙酸共聚物和聚乙二醇等等。
对于第二核酸链，第二核酸链的一端连接于交联核心上，其远离交联核心的一端为粘性末端。第二核酸链的粘性末端与第一核酸链的粘性末端的序列互补，通过碱基互补配对的方式实现交联单元I与支架单元的相互交联。
在一些实施方式中，第二核酸链为单链的核酸链(ssDNA)，第二核酸链的整体作为粘性末端，实现与支架单元的相互交联。在一些实施方式中，第二核酸链也可以是双链的核酸链，并且双链的第二核酸链远离交联核心的一端延伸出ssDNA作为粘性末端。在一些实施方式中，任一第二核酸链均具有相同的核酸序列，且其粘性末端与支架单元中第一核酸链的粘性末端的序列互补。在一些实施方式中，任一第二核酸链的粘性末端均具有相同的核酸序列，且与支架单元中第一核酸链的粘性末端的序列互补。在一些可选的实施方式中，第二核酸链的序列，或者第二核酸链的粘性末端的序列也可以有两种以上的选择，只要对应的支架单元中第一核酸链的粘性末端序列能够实现与其互补即可。
在一些实施方式中，第二核酸链的粘性末端的长度为4nt以上，这样有利于其在生理条件下处于稳定交联状态。优选地，第二核酸链的粘性末端的长度为150nt以下，优选50nt以下，更优选30nt以下，更优选20nt以下。具体的，第二核酸链的粘性末端的长度优选4-30nt，更优选4-20nt。另一方面，第二核酸链的粘性末端的长度也会影响最终交联得到的核酸水凝胶的力学强度，通过设计粘性末端的长度在4-20nt的范围内，可以在实现核酸水凝胶自组装的情况下提高核酸水凝胶的力学强度。
<交联单元II>
交联单元II包括至少2个重复片段，所述重复片段包括单链的第三核酸链和双链的第四核酸链，所述交联单元II由所述第三核酸链和所述第四核酸链交替连接形成，且所述第三核酸链与所述第一核酸链的粘性末端的序列互补。具体地，每个重复片段包括1个单链的第三核酸链和1个双链的第四核酸链，第三核酸链和第四核酸链交替连接，得到由至少2个第三核酸链和至少2个第四核酸链交替连接形成的交联单元II。示例性的，第三核酸链的个数可以是至少2个、至少10个、至少50个、至少100个、至少200个、至少300个、至少500个、至少800个、至少1000个、至少1200个、至少1500个等等，第四核酸链的个数可以是至少2个、至少10个、至少50个、至少100个、至少200个、至少300个、至少500个、至少800个、至少1000个、至少1200个、至少1500个等等。本公开对第三核酸链的个数以及第四核酸链的个数不进行穷举，只要是适于将支架单元和交联单元I的核酸水凝胶结构协同地连接起来，使核酸水凝胶既具有提高的力学性能，又可保持超分子水凝胶动态特性的数量即可。
在一些实施方式中，第三核酸链的个数为至少100个，第四核酸链的个数为至少100个。通过对第三核酸链和对应第四核酸链的个数进行调节，可以实现对交联单元II所提供的交联位点的个数的调节，进而实现对核酸水凝胶的网络结构及结构稳定性的调节。进一步的，交联单元II中，第三核酸链为单链的核酸链，第四核酸链为双链的核酸链，也即，交联单元II是以双链区和单链区交替连接形成的长链的交联单元。其中，第三核酸链的序列与支架单元中第一核酸链的粘性末端的序列互补，提供交联单元II与支架单元的交联位点。通过这种分子设计方式，不仅可以在实现将支架单元协同连接起来，还可以避免交联单元II中的序列内的自组装，以进一步提高核酸水凝胶的结构稳定性，有效抑制核酸链间滑动，得到兼具力学强度和超分子水凝胶动态特性的核酸水凝胶。
在一些优选地实施方式中，交联单元II为延展的长链核酸链。交联单元II的延展性结构更适于与支架单链交联，实现对支架单元和交联单元I交联形成的水凝胶结构的协同连接，更好的抑制核酸链之间的滑动，弥补互补粘性末端的缺口带来的不稳定性。
在本公开中，形成第三核酸链或者第四核酸链分别可以是由脱氧核糖核苷酸和核糖核苷酸中的一种或两种聚合形成。并且，对于组成第三核酸链或者第四核酸链的任意一个核苷酸，可以是修饰的或者是未修饰的核苷酸。
在本公开中，第三核酸链或者第四核酸链的核酸结构可以呈现不同的空间构象。示例性的，核酸可以是L-核酸或D-核酸。
在一些实施方式中，第三核酸链是由脱氧核糖核苷酸形成的单链DNA；在一些实施方式中，第四核酸链是由脱氧核糖核苷酸形成的双链DNA。
在一些实施方式中，第三核酸链的长度为4nt以上，优选地，第三核酸链的长度为150nt以下，优选50nt以下，更优选30nt以下，更优选20nt以下。具体的，第三核酸链的长度优选4-30nt，更优选4-20nt。在一些实施方式中，第四核酸链的长度为4nt以上，优选10-40nt，更优选为20-30nt。
在一些实施方式中，以所述交联单元I和所述交联单元II中重复片段的总摩尔数计，所述重复片段的含量为0.5-99％；可选地，所述重复片段的含量为1-20％；优选地，所述重复片段的含量为2.5-10％。示例性的，所述重复片段的含量为2.5％、5％、7.5％、10％、12％、15％、17％、20％等等。通过调节重复片段的加入比例，能够可控地实现对核酸水凝胶的力学模量的调控。当重复片段的含量为2.5-10％时，随着重复片段的含量的提高，核酸水凝胶的存储模量G’相应提高，机械强度增强。
第二方面
本公开的第二方面提供根据本公开第一方面所述的核酸水凝胶的制备方法，使支架单元与交联单元I和交联单元II交联成型的步骤。
在一些实施方式中，核酸水凝胶的制备方法包括如下步骤：
在凝胶基质中制备支架单元，得到支架单元溶液；
在凝胶基质中制备交联单元I和交联单元II，得到交联单元溶液；
将所述支架单元溶液与所述交联单元溶液混合，所述支架单元与交联单元I和交联单元II自组装，得到核酸水凝胶。
<制备支架单元>
在一些实施方式中，支架单元中的支架核心为核酸，制备支架单元可以通过核酸片段互补配对的方式得到。示例性的，支架单元是以核酸为支架核心，在支架核心上结合三个第一核酸链得到。对应此结构，涉及三个短链的核酸片段，并将其命名为Y1、Y2和Y3。
核酸片段Y1包括顺次连接的单链区Y11、单链区Y12和单链区Y13，核酸片段Y2包括顺次连接的单链区Y21、单链区Y22和单链区Y23，核酸片段Y3包括顺次连接的单链区Y31、单链区Y32和单链区Y33。
其中，单链区Y12的序列与单链区Y33的序列为互补序列，单链区Y13的序列与单链区Y22的序列为互补序列，单链区Y23的序列与单链区Y32的序列为互补序列。
将核酸片段Y1、核酸片段Y2和核酸片段Y3置于同一反应体系中，进行退火处理后，可以得到具有三个第一核酸链的支架单元。在支架单元中，核酸片段Y1的单链区Y12与核酸片段Y3的单链区Y33互补配对为支架双链区，核酸片段Y1的单链区Y13与核酸片段Y2的单链区Y22互补配对为支架双链区，核酸片段Y2的单链区Y23与核酸片段Y3的单链区Y32互补配对为支架双链区，共同构成支架单元的支架核心。并且，单链区Y11、单链区Y21和单链区Y31分别作为连接于支架核心上的三个第一核酸链。
对于凝胶基质，可以是水性介质。水性介质可以是水，或者是以水为溶剂的缓冲液。在一些具体的实施方式中，凝胶基质为磷酸盐缓冲液，磷酸盐缓冲液中包含10mM磷酸盐，100mM NaCl，pH＝7.4。此外，凝胶基质还可以是能够形成核酸水凝胶的其他任意条件的水性介质，本公开对此不作具体限定。
对于退火处理，是将核酸片段Y1、核酸片段Y2和核酸片段Y3孵育后，降温，至核酸片段Y1、核酸片段Y2和核酸片段Y3互补配对形成具有双链区的支架单元。其中，孵育温度、孵育时间，以及降温速度由核酸片段Y1、核酸片段Y2和核酸片段Y3的具体序列结构决定，只要使三者可通过杂交配对的方式，形成支架单元即可。
在一些实施方式中，所述支架核心为多肽，所述支架单元的制备通过将所述第一核酸链与所述作为支架核心的多肽以共价结合的方式进行。优选地所述支架单元通过点击反应，更优选通过铜催化的点击反应制备。
在一些实施方式中，其中所述支架核心为纳米颗粒，所述支架单元的制备通过将所述第一核酸链与作为支架核心的纳米颗粒以共价结合的方式进行。
<制备交联单元I>
在一些实施方式中，交联单元I中的交联核心为核酸，制备交联单元可以通过核酸片段互补配对的方式得到。示例性的，交联单元是以核酸为交联核心，在交联核心上结合2个核酸链得到。对应此结构，涉及2个短链的核酸片段，并将其命名为L1和L2。
核酸片段L1包括顺次连接的单链区L11和单链区L12，核酸片段L2包括顺次连接的单链区L21和单链区L22。其中，单链区L12的序列和单链区L22的序列为互补序列，并且单链区L11的序列与支架单元中单链区Y11、单链区Y21和单链区Y31的至少一个为互补序列，单链区L12的序列与支架单元中单链区Y11、单链区Y21和单链区Y31的至少一个为互补序列。通过单链区L11、单链区L12与单链区Y11、单链区Y21和单链区Y31的序列互补配对，能够实现支架单元和交联单元I的相互交联。
在一些实施方式中，单链区L11的序列与单链区L12的序列相同，单链区Y11、单 链区Y21和单链区Y31的序列相同，并且单链区L11、L12的序列与单链区Y11、Y12、Y13的序列相互补。
将核酸片段L1和核酸片段L2置于同一反应体系中，进行退火处理后，可以得到具有两个第二核酸链的交联单元I。在交联单元I中，核酸片段L1的单链区L22与核酸片段L2的单链区L22互补配对为双链区，并且，单链区L11和单链区L21成为双链区远离连接端的向外延伸的粘性末端，以单链区L11和单链区L21分别作为连接于交联核心上的2个第二核酸链。
在一些更为具体的实施方式中，单链区L12和单链区L22中分别设置有酶切位点，使交联单元I能够被相应的酶切断，从而实现核酸水凝胶特异的酶响应。
对于凝胶基质，可以是水性介质。水性介质可以是水，或者是以水为溶剂的缓冲液。在一些具体的实施方式中，凝胶基质为磷酸盐缓冲液，磷酸盐缓冲液中包含10mM磷酸盐，100mM NaCl，pH＝7.4。此外，凝胶基质还可以是能够形成核酸水凝胶的其他任意条件的水性介质，本公开对此不作具体限定。
对于退火处理，是将核酸片段L1和核酸片段L2孵育后，降温，至核酸片段L1和核酸片段L2互补配对形成具有双链区的支架单元。其中，孵育温度、孵育时间，以及降温速度由核酸片段L1和核酸片段L2的具体序列结构决定，只要使两者可通过杂交配对的方式，形成交联单元I即可。
在一些实施方式中，所述交联核心为多肽，所述交联单元I的制备通过将所述第二核酸链与所述作为交联核心的多肽以共价结合的方式进行。优选地所述交联单元通过点击反应，更优选通过铜催化的点击反应制备。
在一些实施方式中，其中所述交联核心为纳米颗粒，所述交联单元的制备通过将所述第二核酸链与作为交联核心的纳米颗粒以共价结合的方式进行。
<交联单元II>
在一些实施方式中，制备所述交联单元II的步骤包括：
制备由单链区N1和单链区N2交替连接的长链的单链核酸链，所述长链的单链核酸链包括至少2个单链区N1和至少2个单链区N2；
所述长链的单链核酸链与互补链在凝胶基质中混合，退火处理，所述互补链与所述单链区N1的序列互补，形成双链的第四核酸链，所述单链区N2形成第三核酸链，得到第四核酸链和第三核酸链交替连接的交联单元II；其中，相邻的1个单链的第三核酸链和1个双链的第四核酸链连接组成1个重复片段，交联单元II中至少包括2个重复片段。
对于长链的单链核酸链，可以采用本领域中实现长链核酸制备的方法，只要能够得到以单链区N1和单链区N2交替连接形成的长链的单链核酸链即可。在一些实施方式中，长链的单链核酸链是以环状的核酸链为模板，通过滚环扩增得到。在一些实施方式中，长链的单链核酸链是通过化学合成得到。
在一些实施方式中，在焦磷酸酶存在的环境下，制备长链的单链核酸链。具体地，在滚环扩增的反应体系中加入无机焦磷酸酶，通过加入无机焦磷酸酶，用于分解副产物焦磷酸根，避免形成DNA/MgP 2O 7纳米结构。进一步避免以长链的单链核酸链制备的交联单元II发生团聚，使交联单元II的结构尽量铺展，有利于对支架单元的串联连接。
对于环状的核酸链，可以是由线性模板链首尾连接后得到。在一些实施方式中，线性模板链包括2个单链区M1和2个单链区M2，单链区M1和单链区M2交替连接，形成线性的模板链，对线性的模板链进行环化处理，使其连接为环状的核酸链。
对于滚环扩增，是以单链区M1和单链区M2交替排布的环状的核酸链为模板，加入滚换扩增引物，经扩增反应后，得到由大量单链区N1和单链区N2交替连接形成的长链的单链核酸链。其中，单链区N1的序列与单链区M1的序列为互补序列，单链区N2的序列与单链区M2的序列为互补序列。并且，单链区N2的序列与支架单元中单链区 Y11、单链区Y21和单链区Y31的至少一个为互补序列。
在一些实施方式中，单链区N2的序列与交联单元I中单链区L11的序列相同；在一些实施方式中，单链区N2的序列与交联单元I中单链区L21的序列相同；在一些实施方式中，单链区N2的序列与单链区L11、单链区L21的序列均相同；且支架单元中单链区Y11、单链区Y21和单链区Y31的序列相同，单链区N2、L11、L21的序列与单链区Y11、Y21、Y31的序列相互补。
对于退火处理，是将长链的单链核酸链与互补链孵育后，降温，至互补链和单链区N1的序列互补配对形成双链的第四核酸链，单链区N2对应形成单链的第三核酸链。其中，孵育温度、孵育时间，以及降温速度由单链区N1和互补链的具体序列结构决定，只要使两者可通过杂交配对的方式，形成第四核酸链即可。
对于凝胶基质，可以是水性介质。水性介质可以是水，或者是以水为溶剂的缓冲液。在一些具体的实施方式中，凝胶基质为磷酸盐缓冲液，磷酸盐缓冲液中包含10mM磷酸盐，100mM NaCl，pH＝7.4。此外，凝胶基质还可以是能够形成核酸水凝胶的其他任意条件的水性介质，本公开对此不作具体限定。
<支架单元、交联单元I、交联单元II自组装>
将所述支架单元溶液与所述交联单元溶液混合，所述支架单元分别与交联单元I和交联单元II自组装，得到核酸水凝胶。
在一些实施方式中，支架单元溶液是将能够装配形成支架单元的核酸片段溶于凝胶基质后，经退火处理，得到的包含有支架单元的支架单元溶液。
在一些实施方式中，交联单元溶液是将长链的单链核酸链、互补链，与能够装配形成交联单元I的核酸片段共同溶解于凝胶基质中，经退火处理，得到的包含有交联单元I和交联单元II的交联单元溶液。
在一些实施方式中，支架单元溶液与交联单元溶液在4-50℃，优选5-40℃，更优选10-30℃进行混合，使支架单元分别与交联单元I、交联单元II进行碱基互补配对，实现核酸水凝胶的自组装。
在一些实施方式中，支架单元溶液与交联单元溶液在pH 3-11，优选pH 4-10，更优选pH 5-9，还更优选pH 6-8进行混合。
在一些实施方式中，支架单元溶液中的支架单元，与交联单元溶液中总的交联单元的摩尔比为2:1-1:3，优选1:1-1:2，更优选1:1。
以本公开中的方法制备核酸水凝胶，核酸水凝胶可快速成型，具有制备步骤简单，条件易于实现等优势，可以实现核酸水凝胶的快速制备。
第三方面
本公开的第三方面提供了第一方面所述的核酸水凝胶，或第二方面所述的方法制备的核酸水凝胶在如下(a)-(c)至少一种中的用途：
(a)作为或制备生物医用材料；
(b)作为或制备柔性电子材料；
(c)作为或制备三维打印材料。
由于核酸水凝胶的力学强度高、稳定性好，并且兼具超分子水凝胶的动态特性，适合作为生物医用材料用于药物递送、细胞培养分化、蛋白生产、免疫调控等领域中，或者作为柔性电子材料用于可穿戴设备、人造皮肤、软机器人等领域中，或者是作为三维打印材料用于3D打印领域中。
实施例
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限定本公开的范围。实施例中未注明具 体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售获得的常规产品。
本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法，例如下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。
实验材料
下述实施例所采用的实验试剂信息见表1，所设计的DNA序列见表2和序列表。
表1
Figure PCTCN2022086336-appb-000001
表2
Figure PCTCN2022086336-appb-000002
Figure PCTCN2022086336-appb-000003
实施例中DNA序列(包括Y1、Y2、Y3、L1、L2、滚环扩增引物和长链的单链DNA的互补链)采用标准的亚磷酰胺DNA固相合成法(Mermade-12DNA合成仪，美国，Bio Automation公司)合成，并经反相高效液相色谱(Agilent 1200，美国，Agilent公司)分离纯化得到，原料的纯度通过LC-MS(Shimadzu 2020，Japan)表征。实施例中的线性模板链在5’末端有磷酸基团修饰，购买自擎科新业生物技术有限公司(北京，中国)，采用HPLC纯化。所有实验用水均采用18.2MΩ·cm密理博公司生产的超纯水。其他化学试剂均采用优级纯及以上。
实施例1核酸水凝胶的制备
步骤1，使用滚环扩增反应来制备长链的单链DNA，具体如下：
将350pmol的线性模板链、10μL的10×ssDNA/RNA环化连接酶反应缓冲液、3μL的ssDNA/RNA环化连接酶、5μL的50mM MnCl 2混合均匀，加入适量高纯水至总体积为100μL。在60℃下孵育6h进行环化反应，再在80℃下反应10min灭活连接酶，室温下放置10min以使其冷却至室温，将得到环状的模板DNA。
向上述体系中加入5μL核酸外切酶Ⅰ和2.5μL核酸外切酶Ⅲ。在37℃下反应30min将未环化的线性模板链消化，然后90℃灭活5min，在室温条件下冷却10min。反应产物于20％丙烯酰胺/8M尿素变性凝胶电泳后，观察环化或连接产物情况。利用截留分子量为3000Da的超滤管超滤3次进行纯化。
将表3所示的各组分混合后于30℃下孵育4h，随后于65℃孵育10min将酶失活。反应体系中加入无机焦磷酸酶，用于分解副产物焦磷酸根，避免形成DNA/MgP 2O 7纳米结构，从而得到游离的长链的单链DNA。
表3
组分 体积(μl) 100μl体系终浓度
Phi29 DNA聚合酶反应缓冲液 10 1×
BSA 1 200μg/mL
dNTPs 10 1mM
环状模板链 60 500nM
滚环扩增引物 1.5 1μM
phi29 DNA聚合酶 5 0.2U/μl
无机焦磷酸酶 5 0.005U/μl
超纯水 2.5  
使用紫外分光光度计(Nanodrop 2000，Thermo Scientific)对长链的单链DNA在260nm处的吸光度进行测定。使用重复片段序列在260nm处的消光系数计算长链的单链 DNA中所有重复序列的量。
步骤2，各取20nmol的Y1、Y2和Y3混合，水溶液置于离心管中，冷冻干燥。向冻干产物中加入20μl磷酸缓冲液(PBS，10mM磷酸盐，100mM NaCl，pH＝7.4)中，95℃加热5min，自然冷却至室温，得到支架单元溶液。
步骤3，各29.25nmol的L1、L2、含有1.5nmol重复片段的长链的单链DNA和1.5nmol长链的单链DNA的互补链混合，互补链的量与长链的单链DNA重复序列量相等，L1、L2与长链的单链DNA重复序列的总量为60nmol。冷冻干燥后加入20μl磷酸缓冲液，95℃加热5min，自然冷却至室温，得到交联单元II中重复片段的摩尔含量为2.5％的交联单元溶液。
步骤4，将支架单元溶液和交联单元溶液在室温下混合，震荡离心后得到不同交联单元II含量的DNA超分子水凝胶，也即核酸水凝胶。
实施例2核酸水凝胶的制备
以实施例1中步骤1-步骤4所示的方法制备核酸水凝胶，其中，步骤3中交联单元溶液的制备如下所示：
各取28.50nmol的L1和L2，含有3.0nmol重复序列的长链的单链DNA溶液和3.0nmol的互补链混合后冷冻干燥，得到交联单元II中重复片段的摩尔含量为5.0％的交联单元溶液。
实施例3核酸水凝胶的制备
以实施例1中步骤1-步骤4所示的方法制备核酸水凝胶，其中，步骤3中交联单元溶液的制备如下所示：
各取27.75nmol的L1和L2，含有4.5nmol重复序列的长链的单链DNA溶液和4.5nmol的互补链混合后冷冻干燥，得到交联单元II中重复片段的摩尔含量为7.5％的交联单元溶液。
实施例4核酸水凝胶的制备
以实施例1中步骤1-步骤4所示的方法制备核酸水凝胶，其中，步骤3中交联单元溶液的制备如下所示：
各取27.00nmol的L1和L2，含有6.0nmol重复序列的长链的单链DNA溶液和6.0nmol的互补链混合后冷冻干燥，得到交联单元II中重复片段的摩尔含量为10.0％的交联单元溶液。
对比例1
以实施例1中步骤2-步骤4所示的方法制备核酸水凝胶，其中，步骤3中交联单元溶液的制备如下所示：
各取30nmol的L1和L2混合后冷冻干燥，得到不含有交联单元II的交联单元溶液。
性能测试
采用英国马尔文公司生产的Kinexus Pro+旋转流变仪测试各水凝胶样品的流变学性质。选用直径为8mm的平行板转子进行测试，设定测试台与平行板的间距为150μm。将40μL水凝胶样品置于测试台中心，转子下压后，四周多余的凝胶用药匙轻轻刮去，并在四周滴加少量硅油密封以阻止水分挥发。
固定温度为25℃，应变振幅为1％，振荡频率为1Hz，测试实施例1-4和对比例1中的核酸水凝胶的储能模量G’和损耗模量G”。实施例1-4和对比例1各测试三个平行样品，统计后结果如图2所示。储存模量G’反应了材料的弹性，即刚性，储存模量越大，材料越不容易变形。从图2中可以看出，各实施例的核酸水凝胶的储能模量G’均大于对比例水凝胶的G’，且随着交联单元II中重复片段含量的提升，核酸水凝胶的G’逐渐升高。说明核酸水凝胶的力学性能得到有效提升。
由于超分子相互作用的可逆性和动态性，力学增强的核酸水凝胶具有自修复特性。 两块独立制备的实施例2水凝胶接触后会粘附在一起，室温下放置一段时间后两块水凝胶会逐渐融合，没有明显界面存在。使用流变仪对自愈合前后的水凝胶的流变性质进行测量，在25℃，1％应变振幅，1Hz振荡频率时的力学模量如图3所示，自愈合前后水凝胶的力学模量没有明显差异。
力学增强DNA水凝胶同时保留有超分子水凝胶剪切变稀和可注射特性。如图4所示，实施例2水凝胶在注射前在注射器中为凝胶态，注射时受针头内壁剪切力的作用，粘度下降，可以顺利通过针头。注射结束后样品快速回复到凝胶状态，形成纤维状凝胶细丝。
本公开的上述实施例仅是为清楚地说明本公开所作的举例，而并非是对本公开的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本公开的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本公开权利要求的保护范围之内。

Claims (11)

  1. 一种核酸水凝胶，其中，所述核酸水凝胶包括支架单元，以及分别与所述支架单元相交联的交联单元I和交联单元II；
    所述支架单元包括支架核心和与所述支架核心结合的至少3个第一核酸链，所述交联单元I包括交联核心和与所述交联核心结合的至少2个第二核酸链；所述第一核酸链远离所述支架核心的一端为粘性末端，且与所述第二核酸链远离所述交联核心的粘性末端的序列互补；
    所述交联单元II包括至少2个重复片段，所述重复片段包括单链的第三核酸链和双链的第四核酸链，所述交联单元II由所述第三核酸链和所述第四核酸链交替连接形成，且所述第三核酸链与所述第一核酸链的粘性末端的序列互补。
  2. 根据权利要求1所述的核酸水凝胶，其中，以所述交联单元I和所述交联单元II中重复片段的总摩尔数计，所述重复片段的含量为0.5-99％；可选地，所述重复片段的含量为1-20％；优选地，所述重复片段的含量为2.5-10％。
  3. 根据权利要求1或2所述的核酸水凝胶，其中，形成所述支架核心或所述交联核心的材料彼此独立地选自由如下材料组成的组：核酸、多肽、高分子化合物和纳米颗粒。
  4. 根据权利要求1-3任一项所述的核酸水凝胶，其中，所述第一核酸链、第二核酸链、第三核酸链和第四核酸链中的任一核酸链的任一核苷酸为修饰的核苷酸或未修饰的核苷酸；
    可选地，所述第一核酸链、第二核酸链、第三核酸链和第四核酸链中的任一核酸链的任一核苷酸为脱氧核糖核苷酸或核糖核苷酸。
  5. 根据权利要求1-4任一项所述的核酸水凝胶，其中，所述第一核酸链、第二核酸链中的任一核酸链的粘性末端的长度为4nt以上，优选4-30nt，更优选4-20nt；
    可选地，所述第三核酸链的长度为4nt以上，优选4-30nt，更优选4-20nt。
  6. 根据权利要求1-5任一项所述的核酸水凝胶，其中，所述第四核酸链的长度为4nt以上，优选10-40nt，更优选为20-30nt。
  7. 一种制备根据权利要求1-6任一项所述的核酸水凝胶的方法，其中，所述方法包括使支架单元与交联单元I和交联单元II交联成型的步骤。
  8. 根据权利要求7所述的方法，其中，所述方法包括如下步骤：
    在凝胶基质中制备支架单元，得到支架单元溶液；
    在凝胶基质中制备交联单元I和交联单元II，得到交联单元溶液；
    将所述支架单元溶液与所述交联单元溶液混合，所述支架单元与交联单元I和交联单元II自组装，得到核酸水凝胶。
  9. 根据权利要求8所述的方法，其中，制备所述交联单元II的步骤包括：
    制备由单链区N1和单链区N2交替连接的长链的单链核酸链，所述长链的单链核酸链包括至少2个单链区N1和至少2个单链区N2；
    所述长链的单链核酸链与互补链在凝胶基质中混合，退火处理，所述互补链与所述单链区N1的序列互补，形成双链的第四核酸链，所述单链区N2形成第三核酸链，得到第四核酸链和第三核酸链交替连接的交联单元II；
    可选地，以环状的核酸链为模板，通过滚环扩增，制备长链的单链核酸链；
    优选地，在焦磷酸酶存在的环境下，制备长链的单链核酸链。
  10. 一种制备核酸水凝胶的方法，其中，包括以下步骤：
    制备支架单元，所述支架单元包括支架核心和与所述支架核心结合的至少3个第一核酸链；
    制备交联单元I，所述交联单元I包括交联核心和与所述交联核心结合的至少2个第二核酸链；
    制备交联单元II，所述交联单元II由长链的单链核酸链与互补链的序列互补形成；所 述交联单元II包括至少2个重复片段，所述重复片段包括单链的第三核酸链和双链的第四核酸链，所述交联单元II由所述第三核酸链和所述第四核酸链交替连接形成；
    其中，所述第一核酸链远离所述支架核心的一端为粘性末端，且与所述第二核酸链远离所述交联核心的粘性末端的序列互补；所述第三核酸链与所述第一核酸链的粘性末端的序列互补；
    在凝胶基质中，所述支架单元分别与所述交联单元I和所述交联单元II相交联，得到核酸水凝胶。
  11. 一种权利要求1-6任一项所述的核酸水凝胶，或以权利要求7-10任一项所述的方法制备的核酸水凝胶在如下(a)-(c)至少一种中的用途：
    (a)作为或制备生物医用材料；
    (b)作为或制备柔性电子材料；
    (c)作为或制备三维打印材料。
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