CN113083172A

CN113083172A - Nucleic acid hydrogel with improved mechanical properties and preparation method and application thereof

Info

Publication number: CN113083172A
Application number: CN202110395102.8A
Authority: CN
Inventors: 刘冬生; 李宇杰; 杨勃
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-04-13
Filing date: 2021-04-13
Publication date: 2021-07-09
Anticipated expiration: 2041-04-13
Also published as: WO2022218302A9; WO2022218302A1; CN113083172B

Abstract

The disclosure belongs to the technical field of high polymer materials and biology, and particularly relates to a nucleic acid hydrogel with improved mechanical properties, and a preparation method and application thereof. The nucleic acid hydrogel is formed by respectively intersecting and linking a bracket unit with a crosslinking unit I and a crosslinking unit II, and has improved mechanical strength and stability. Meanwhile, the nucleic acid hydrogel keeps the dynamic characteristics of the supramolecular hydrogel, has the performances of rapid forming, shear thinning, injectability, self-repairability, thermoplasticity and the like, has good biocompatibility, and has important application prospects in biomedical fields of drug delivery, cell culture differentiation, protein production, immune regulation and the like, and flexible electronic fields of wearable equipment, artificial skin, soft robots and the like.

Description

Nucleic acid hydrogel with improved mechanical properties and preparation method and application thereof

Technical Field

The disclosure belongs to the technical field of high polymer materials and biology, and particularly relates to a nucleic acid hydrogel and a preparation method and application thereof.

Background

The supermolecular hydrogel is a soft material with a three-dimensional network structure formed by self-assembly based on non-covalent interaction. Compared with the covalently crosslinked hydrogel, the transient reversibility of non-covalent interaction endows the supramolecular hydrogel with good dynamic characteristics, such as stimulation responsiveness, injectability, shape adaptability, self-repairing performance and the like, and has great application potential in numerous fields of biomedicine, flexible electronic materials, soft robots and the like. However, the rapid dissociation and recombination rates of supramolecular interactions make supramolecular hydrogels with poor mechanical properties, which limits the applications of supramolecular hydrogels as structural materials in many fields. Therefore, it has been a long-standing challenge to design and prepare a hydrogel having both high mechanical strength and dynamic properties.

At present, various methods can be used for enhancing the mechanical strength of the supramolecular hydrogel, including methods of introducing a covalent cross-linking network, doping a nano composite material and the like. The introduction of the covalent cross-linked network can effectively dissipate energy by constructing a flexible and rigid interconnected network, thereby ensuring the excellent mechanical properties of the hydrogel. However, due to the irreversibility of the covalent crosslinking sites, the dynamic properties of the hydrogel will disappear, and in addition, when the covalent crosslinking network is broken, permanent damage and poor fatigue resistance of the hydrogel will result. The nano materials, such as carbon nanoparticles, quantum dots, metal nanoparticles and the like, can also be introduced into the supramolecular hydrogel to serve as additional crosslinking points, so that the mechanical property of the hydrogel is improved, and the supramolecular hydrogel can also have additional physical properties. But the introduction of nanomaterials may bring unknown biotoxicity. In addition, the methods can change the crosslinking structure and the crosslinking density of the hydrogel network while enhancing the mechanical property, and the original topological structure, mesh distribution and permeability of the supramolecular hydrogel network can not be retained.

Nucleic acid molecules can encode, store and transmit genetic information and are one of the core molecules of living systems. In recent years, with the rapid development of chemistry, materials science, and nanotechnology, not only a great deal of research and research has been conducted on nucleic acid molecules as genetic information carriers, but also they have been used as a kind of assembly material. From the material and chemical perspectives, nucleic acid molecules have the characteristics of definite structure, specific recognition of bases, easy functional modification and the like, and are widely concerned and developed in the fields of chemistry, biology, materials science and the like. At present, people have designed and synthesized various nucleic acid nano materials by utilizing the excellent performance of nucleic acid molecules. The nucleic acid hydrogel is a typical representative, the nucleic acid molecules are utilized to prepare the hydrogel, the skeleton function of the hydrogel can be utilized, the biological function of DNA can also be utilized, the unified fusion of the structure and the function of the hydrogel material is realized, and the nucleic acid hydrogel has wide application in a plurality of biomedical fields including drug delivery and slow release, biological detection, protein production, immune regulation and the like.

The nucleic acid hydrogel has a wide application prospect in multiple fields, and the important problem to be solved in the field is how to further improve the mechanical property of the nucleic acid hydrogel, and the dynamic property of the supramolecular hydrogel is achieved while the mechanical strength of the nucleic acid hydrogel is improved.

Disclosure of Invention

Problems to be solved by the invention

In view of the problems in the prior art, for example, it is required to improve the mechanical properties of nucleic acid hydrogel and to realize the dynamic properties of nucleic acid hydrogel such as injectability and self-healing. Therefore, the nucleic acid hydrogel is formed by respectively cross-linking the support unit with the cross-linking unit I and the cross-linking unit II, has the advantages of high mechanical strength and good stability through the cooperative cooperation of the support unit, the cross-linking unit I and the cross-linking unit II, can keep the dynamic characteristics of the supermolecule hydrogel while keeping the full rigid network structure of the nucleic acid hydrogel, and has important application prospects in the fields of biomedicine, flexible electronics and the like.

Means for solving the problems

The present disclosure first provides a nucleic acid hydrogel, wherein the nucleic acid hydrogel comprises a scaffold unit, and a crosslinking unit I and a crosslinking unit II that are crosslinked with the scaffold unit, respectively;

the scaffold unit comprises a scaffold core and at least 3 first nucleic acid strands bound to the scaffold core, the cross-linking unit I comprises a cross-linking core and at least 2 second nucleic acid strands bound to the cross-linking core; the end of the first nucleic acid strand distal to the scaffold core is a cohesive end and is complementary to the sequence of the second nucleic acid strand distal to the cohesive end of the cross-linked core;

the crosslinking unit II comprises at least 2 repeating segments comprising a single-stranded third nucleic acid strand and a double-stranded fourth nucleic acid strand, the crosslinking unit II is formed by alternately connecting the third nucleic acid strand and the fourth nucleic acid strand, and the third nucleic acid strand is complementary to a sequence of a cohesive end of the first nucleic acid strand.

In some embodiments, the nucleic acid hydrogel according to the present disclosure, wherein the content of the repeating segments is 0.5 to 99% based on the total number of moles of the repeating segments in the crosslinking unit I and the crosslinking unit II; optionally, the content of the repetitive fragment is 1-20%; preferably, the content of the repetitive fragment is 2.5-10%.

In some embodiments, 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.

In some embodiments, the nucleic acid hydrogel according to the present disclosure, wherein any nucleotide of any one of the first, second, third and fourth nucleic acid strands is a modified nucleotide or an unmodified nucleotide;

optionally, any nucleotide of any one of the first, second, third and fourth nucleic acid strands is a deoxyribonucleotide or a ribonucleotide.

In some embodiments, the nucleic acid hydrogel according to the present disclosure, wherein the length of the cohesive end of any one of the first nucleic acid strand and the second nucleic acid strand is 4nt or more, preferably 4 to 30nt, more preferably 4 to 20 nt;

alternatively, the length of the third nucleic acid strand is 4nt or more, preferably 4 to 30nt, more preferably 4 to 20 nt.

In some embodiments, the nucleic acid hydrogel according to the present disclosure, wherein the length of the fourth nucleic acid strand is 4nt or more, preferably 10 to 40nt, more preferably 20 to 30 nt.

The present disclosure also provides a method for preparing the nucleic acid hydrogel according to the present disclosure, wherein the method comprises a step of crosslinking and molding the scaffold unit with the crosslinking unit I and the crosslinking unit II.

In some embodiments, the method according to the present disclosure, wherein the method comprises the steps of:

preparing a scaffold unit in a gel matrix to obtain a scaffold unit solution;

preparing a crosslinking unit I and a crosslinking unit II in a gel matrix to obtain a crosslinking unit solution;

and mixing the scaffold unit solution with the crosslinking unit solution, and self-assembling the scaffold unit with a crosslinking unit I and a crosslinking unit II to obtain the nucleic acid hydrogel.

In some embodiments, the method according to the present disclosure, wherein the step of preparing the crosslinking unit II comprises:

preparing a long-chain single-stranded nucleic acid strand comprising at least 2 single-stranded regions N1 and at least 2 single-stranded regions N2 alternately connected by single-stranded regions N1 and N2;

mixing the long-chain single-stranded nucleic acid chain with a complementary chain in a gel matrix, and annealing, wherein the complementary chain is complementary to the sequence of the single-stranded region N1 to form a double-stranded fourth nucleic acid chain, and the single-stranded region N2 forms a third nucleic acid chain, so that a cross-linking unit II which is alternately connected with the fourth nucleic acid chain and the third nucleic acid chain is obtained;

optionally, preparing a long-chain single-stranded nucleic acid chain by rolling circle amplification with a circular nucleic acid chain as a template;

preferably, the long-chain single-stranded nucleic acid strand is prepared in the presence of pyrophosphatase.

The present disclosure also provides a method of preparing a nucleic acid hydrogel, comprising the steps of:

preparing a scaffold unit comprising a scaffold core and at least 3 first nucleic acid strands bound to the scaffold core;

preparing a cross-linking unit I comprising a cross-linked core and at least 2 second nucleic acid strands bound to the cross-linked core;

preparing a crosslinking unit II formed by complementing the sequences of a long-chain single-stranded nucleic acid chain and a complementary chain; the cross-linking unit II comprises at least 2 repeated segments, the repeated segments comprise a third nucleic acid strand which is single-stranded and a fourth nucleic acid strand which is double-stranded, and the cross-linking unit II is formed by the alternating connection of the third nucleic acid strand and the fourth nucleic acid strand;

wherein the end of the first nucleic acid strand distal to the scaffold core is a sticky end and is complementary to the sequence of the second nucleic acid strand distal to the sticky end of the cross-linking core; the third nucleic acid strand is complementary to the sequence of the cohesive end of the first nucleic acid strand.

In the gel matrix, the scaffold unit is respectively crosslinked with the crosslinking unit I and the crosslinking unit II to obtain the nucleic acid hydrogel.

In some embodiments, a method of making a nucleic acid hydrogel according to the present disclosure, wherein long-chain single-stranded nucleic acid strands comprising at least 2 single-stranded regions N1 and at least 2 single-stranded regions N2 are alternately linked by single-stranded regions N1 and single-stranded regions N2;

In some embodiments, the method of preparing a nucleic acid hydrogel according to the present disclosure, wherein the amount of the repeating segments in the crosslinking unit II is 0.5 to 99% based on the total number of moles of the repeating segments in the crosslinking unit I and the crosslinking unit II; optionally, the content of the repeating segments in the crosslinking unit II is 1-20%; preferably, the content of the repeating segment in the crosslinking unit II is 2.5 to 10%.

In some embodiments, the method of preparing a nucleic acid hydrogel according to the present disclosure, wherein the material forming the scaffold core or the cross-linked core is independently selected from the group consisting of: nucleic acids, polypeptides, polymeric compounds, and nanoparticles.

In some embodiments, the method of preparing a nucleic acid hydrogel according to the present disclosure, wherein any nucleotide of any one of the first, second, third and fourth nucleic acid strands is a modified nucleotide or an unmodified nucleotide;

In some embodiments, the method for preparing a nucleic acid hydrogel according to the present disclosure, wherein the length of the cohesive end of any one of the first nucleic acid strand and the second nucleic acid strand is 4nt or more, preferably 4 to 30nt, more preferably 4 to 20 nt;

In some embodiments, the method for preparing a nucleic acid hydrogel according to the present disclosure, wherein the length of the fourth nucleic acid strand is 4nt or more, preferably 10 to 40nt, more preferably 20 to 30 nt.

The present disclosure also provides a nucleic acid hydrogel according to the present disclosure, or a nucleic acid hydrogel prepared by a method according to the present disclosure, for use in at least one of (a) - (c) below:

(a) as or in the preparation of biomedical materials;

(b) as or in the preparation of flexible electronic materials;

(c) as or to prepare a three-dimensional printed material.

ADVANTAGEOUS EFFECTS OF INVENTION

In some embodiments, the nucleic acid hydrogel provided by the present disclosure is formed by cross-linking a scaffold unit with a cross-linking unit I and a cross-linking unit II, respectively, and has a spatial structure of a three-dimensional network, and the cross-linking unit II, the cross-linking unit I, and the scaffold unit are cooperatively constructed, so that effective control of mechanical properties of the nucleic acid hydrogel is achieved, and mechanical strength and stability of the nucleic acid hydrogel are improved. Meanwhile, the nucleic acid hydrogel keeps the dynamic characteristics of the supramolecular hydrogel, has the performances of rapid forming, shear thinning, injectability, self-repairability, thermoplasticity and the like, has good biocompatibility, and has important application prospects in biomedical fields of drug delivery, cell culture differentiation, protein production, immune regulation and the like, and flexible electronic fields of wearable equipment, artificial skin, soft robots and the like.

In some embodiments, the nucleotides composing the first nucleic acid strand, the second nucleic acid strand, the third nucleic acid strand or the fourth nucleic acid strand of the nucleic acid hydrogel provided by the present disclosure may be modified nucleotides, and by introducing the modification of the nucleotides, the stability of the nucleic acid hydrogel can be further improved, the immunogenicity thereof can be improved, and the application range of the nucleic acid hydrogel in the field of biomedicine can be widened.

In some embodiments, the present disclosure provides a method for preparing a nucleic acid hydrogel, which has the advantages of simple preparation steps and capability of realizing rapid formation of the nucleic acid hydrogel.

Drawings

FIG. 1 shows a schematic diagram of a process for preparing a nucleic acid hydrogel, FIG. 1A shows a DNA hydrogel prepared with a scaffold unit and a crosslinking unit I, and FIG. 1B shows a DNA hydrogel with enhanced mechanical properties prepared with a scaffold unit, a crosslinking unit I and a crosslinking unit II;

FIG. 2 shows the results of rheological property tests of nucleic acid hydrogels prepared in various examples and comparative examples of the present disclosure;

FIG. 3 shows a comparison of mechanical properties before and after self-healing of a nucleic acid hydrogel prepared in example 2 of the present disclosure;

fig. 4 shows injectability of the nucleic acid hydrogel prepared in example 2 of the present disclosure.

Detailed Description

Hereinafter, the present disclosure will be described in detail. The technical features described below are explained based on representative embodiments and specific examples of the present disclosure, but the present disclosure is not limited to these embodiments and specific examples.

It should be noted that:

in the present disclosure, the numerical range represented by "numerical value a to numerical value B" means a range including the endpoint numerical value A, B.

In the present disclosure, "more" of "plural", and the like means a numerical value of 2 or more, unless otherwise specified.

In the present disclosure, the term "substantially", "substantially" or "essentially" means an error of less than 5%, or less than 3% or less than 1% compared to the relevant perfect or theoretical standard.

In the present disclosure, "%" denotes mass% unless otherwise specified.

In the present disclosure, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In the present disclosure, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

In this disclosure, although the disclosure supports the definition of the term "or", "or" as merely alternatives and "and/or", the term "or", "or" means "and/or" in the claims unless expressly indicated to be only an alternative or an exclusion of one another.

As used in this disclosure, "water" includes any feasible water such as tap water, deionized water, distilled water, double distilled water, purified water, ion-exchanged water, and the like.

In the present disclosure, modifications of nucleotides include modifications to ribose, modifications to base, and modifications to phosphorusOne or more modifications of the acid diester bond. Exemplary nucleotide modifications may be LNA, 2 '-OMe, 3' -OMeU, vmoe, Phosphorothioate (PS), m⁶A、Ψ、m¹A, and so on.

In the present disclosure, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and are polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

First aspect

A first aspect of the present disclosure provides a nucleic acid hydrogel, wherein the nucleic acid hydrogel comprises a scaffold unit, and a crosslinking unit I and a crosslinking unit II that are crosslinked with the scaffold unit, respectively;

In the current nucleic acid hydrogel, there is a DNA hydrogel formed by crosslinking Branched DNA Monomers (BDM) and Linker DNA, wherein the BDM may be X-type, T-type, and Y-type DNA monomers formed by connecting 3, 4, etc. DNA short arms together, and fig. 1A shows a DNA hydrogel formed by crosslinking a Y-type DNA monomer (Y-DNA, i.e., scaffold unit) and Linker DNA (Linker, i.e., crosslinking unit I). The Y-DNA has 3 short arms connected with each other, dsDNA is arranged in the short arms close to the connection position, and the free end of the dsDNA is provided with a section of ssDNA which is a sticky end. The 5 'end and the 3' end of the adapter DNA are respectively designed with sticky ends which are complementary with the ssDNA sequence of the Y-DNA monomer, and the Y-DNA monomer and the adapter DNA can be cross-linked to form the DNA hydrogel with a three-dimensional network structure in a base complementary pairing mode. The DNA hydrogel obtained by the molecular design improves the controllability of the mechanical property of the nucleic acid hydrogel to a certain extent. However, the Y-DNA monomer and the adapter DNA realize self-assembly by a non-covalent bond mode, the stability of a cross-linking point is insufficient, and the stability and the mechanical strength of the DNA hydrogel need to be further improved.

In the nucleic acid hydrogel of the present disclosure, the cohesive end of the first nucleic acid strand in the scaffold unit is capable of crosslinking the scaffold unit with the crosslinking unit I by being complementary to the sequence of the cohesive end of the second nucleic acid strand in the crosslinking unit I to construct a hydrogel having a three-dimensional network structure. Meanwhile, the cohesive end of the third nucleic acid chain in the cross-linking unit II is complementary to the sequence of the cohesive end of the first nucleic acid chain in the scaffold unit, so that the cross-linking of the cross-linking unit II and the scaffold unit is realized, and the three jointly participate in the construction of the three-dimensional space structure of the nucleic acid hydrogel (FIG. 1B). After the cross-linking unit II is introduced into the hydrogel structure, the scaffold unit and the hydrogel structure formed by cross-linking the cross-linking unit I are equivalently connected in a covalent bond mode, so that the stability of the cross-linking points of the nucleic acid hydrogel is synergistically improved, the sliding among nucleic acid chains can be better inhibited, the instability caused by the gap of the complementary viscous tail end can be compensated, and the nucleic acid hydrogel has higher mechanical strength and stability.

Meanwhile, the nucleic acid hydrogel formed by jointly crosslinking the scaffold unit, the crosslinking unit I and the crosslinking unit II is discovered to have the full-rigid network structure of the nucleic acid hydrogel, and simultaneously retain the dynamic characteristics of the supramolecular hydrogel, such as good shear thinning, injectability, quick self-healing and stimulation response properties.

In addition, in the process of improving the mechanical property and maintaining the dynamic property of the supramolecular hydrogel, the nucleic acid hydrogel disclosed by the disclosure does not need to add guest molecules to construct the double-network hydrogel, and has the characteristics of easiness in preparation and quick response.

< holder Unit >

A scaffold unit in the present disclosure includes a scaffold core and at least 3 first nucleic acid strands bound to the scaffold core. Illustratively, the number of first nucleic acid strands can be 3, 4, 5, and so forth. The first nucleic acid strand is bound to the scaffold core such that the scaffold unit forms a nucleic acid monomer structure around which 3, 4, 5, etc. nucleic acid short arms are attached.

In some embodiments, the number of the first nucleic acid strands is 3, and 3 first nucleic acid strands are bound to the scaffold core to obtain a Y-shaped nucleic acid monomer structure, which is beneficial to constructing a nucleic acid hydrogel with high spatial structure stability.

For the scaffold core, the material forming the scaffold core may be a nucleic acid, a polypeptide, a polymer compound, or a nanoparticle. In addition, the scaffold core may 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.

In some embodiments, the scaffold core is a nucleic acid. The nucleic acid serving as a core of the scaffold in the present disclosure may be formed by polymerizing one or both of deoxyribonucleotides and ribonucleotides. Also, any one of the nucleotides constituting the nucleic acid may be a modified or unmodified nucleotide.

In the present disclosure, the nucleic acids may assume different spatial conformations. Illustratively, the nucleic acid may be an L-nucleic acid or a D-nucleic acid.

In some embodiments, the scaffold core is DNA formed from deoxyribonucleotides. Further, the DNA as a core of the scaffold has a scaffold double-stranded region which is complementary paired, and the number of the scaffold double-stranded regions is set corresponding to the number of the first nucleic acid strands. Both ends of each scaffold double-stranded region are respectively connected with other scaffold double-stranded regions and the first nucleic acid strand, and for convenience of description, one end of the scaffold double-stranded region connected with the other scaffold double-stranded regions is referred to as a connecting end. The scaffold double-stranded regions are connected to each other through the connecting end to form a scaffold core in a Y-type, X-type, or the like structure, and one end of each scaffold double-stranded region remote from the connecting end is connected to one first nucleic acid strand.

In some more specific embodiments, the scaffold double-stranded region has a length of 4bp or more, preferably 4 to 150bp, preferably 5 to 50bp, more preferably 6 to 30bp, and still more preferably 8 to 20 bp.

In some embodiments, the scaffold core is a polypeptide formed by two or more amino acids linked to each other by peptide bonds, and may encompass dipeptides, tripeptides, tetrapeptides, oligopeptides, proteins, and the like.

In some embodiments, the scaffold core is a nanoparticle, which is a microscopic particle on the nanometer scale, generally referring to a particle that is less than 100nm in at least one dimension. In particular, semiconductor nanoparticles smaller than 10nm are also referred to as quantum dots due to their electronic energy level quantization. Nanoparticles as scaffold cores encompass quantum dots, Fe₂O₃、Si、SiO₂Au and Ag nanoparticles, and the like.

In some embodiments, the scaffold core is a macromolecular compound, which is a compound having a relative molecular mass of several thousand to several million, connected by simple structural units in a repetitive manner. Specifically, the high molecular compound as the core of the scaffold encompasses polylactic acid, polylactic acid-glycolic acid copolymer, polyethylene glycol, and the like.

For the first nucleic acid strand, one end of the first nucleic acid strand is attached to the scaffold core and the end thereof remote from the scaffold core is a cohesive 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 to the sequence of the third nucleic acid strand. After the scaffold unit is mixed with the crosslinking unit I and the crosslinking unit II in the gel matrix, the cohesive ends of the first nucleic acid strand can be self-assembled to obtain the nucleic acid hydrogel by complementary base pairing with the cohesive ends of the second nucleic acid strand, and the cohesive ends of the first nucleic acid strand can be complementarily base paired with the cohesive ends of the third nucleic acid strand.

In some embodiments, the first nucleic acid strand is a single-stranded nucleic acid strand (ssDNA) having the entirety of the first nucleic acid strand as a cohesive end effecting cross-linking with each other of cross-linking unit I and cross-linking unit II. In some embodiments, the first nucleic acid strand may also be a double-stranded nucleic acid strand, and the end of the double-stranded first nucleic acid strand distal to the scaffold core extends beyond ssDNA as a sticky end. In some embodiments, any 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 cross-linking unit I, and the sequence of the third nucleic acid strand. In some embodiments, the cohesive ends of any of the first nucleic acid strands have the same nucleic acid sequence and are complementary to the cohesive ends of the second nucleic acid strand in cross-linking unit I, and to the sequence of the third nucleic acid strand. In some alternative embodiments, the sequence of the first nucleic acid strand, or the sequence of the cohesive end of the first nucleic acid strand, may also be chosen from more than two, as long as the sequence of the cohesive end of the second nucleic acid strand, and the sequence of the third nucleic acid strand in the corresponding cross-linking unit I are capable of achieving complementarity thereto. By changing the length or sequence of the cohesive ends, the thermal stability and mechanical strength of the nucleic acid hydrogel can be adjusted, thereby endowing the gel with different temperature responsiveness and mechanical properties.

In some embodiments, the cohesive end of the first nucleic acid strand is 4nt or more in length, which facilitates its stable cross-linked state under physiological conditions. Preferably, the cohesive end of the first nucleic acid strand has a length of 150nt or less, preferably 50nt or less, more preferably 30nt or less, and still more preferably 20nt or less. Specifically, the length of the cohesive end of the first nucleic acid strand is preferably 4 to 30nt, more preferably 4 to 20 nt. On the other hand, the length of the cohesive end of the first nucleic acid strand also affects the mechanical strength of the nucleic acid hydrogel finally obtained by crosslinking, and the mechanical strength of the nucleic acid hydrogel can be improved while achieving self-assembly of the nucleic acid hydrogel by designing the length of the cohesive end to be in the range of 4 to 20 nt.

< crosslinking Unit I >

The crosslinking unit I in the present disclosure includes a crosslinking core and at least 2 second nucleic acid strands bound to the crosslinking core. Illustratively, the number of first nucleic acid strands can be 2, 3, 4, and so forth. The second nucleic acid strand is bound to the crosslinking core such that the crosslinking unit I forms a nucleic acid adaptor structure surrounding the short nucleic acid arm to which 2, 3, 4, etc. are linked.

In some embodiments, the number of the first nucleic acid strands is 2, and 2 first nucleic acid strands are bound to the cross-linking core to form a nucleic acid adaptor structure, which facilitates the construction of a nucleic acid hydrogel with high spatial structure stability.

For the crosslinking unit I, the material forming the crosslinked core may be a nucleic acid, a polypeptide, a polymer compound, or a nanoparticle. In addition, the crosslinked core may also be formed from other types of biomaterials known in the art, so long as they are suitable for use in the construction of nucleic acid hydrogels.

In some embodiments, the crosslinked core is a nucleic acid. The nucleic acid as a crosslinked core in the present disclosure may be formed by polymerization of one or both of deoxyribonucleotides and ribonucleotides. Also, any one of the nucleotides constituting the nucleic acid may be a modified or unmodified nucleotide.

In some embodiments, the crosslinked core is DNA formed from deoxyribonucleotides. Further, the DNA as a crosslinking core has a complementary paired crosslinked double-stranded region, and both ends or one end of the crosslinked double-stranded region are linked to a second nucleic acid strand to form a crosslinking unit I. The number of cross-linked double-stranded regions can be 1, 2, 3, etc. Illustratively, the number of the cross-linked double-stranded regions is 1, and a second nucleic acid strand is connected to each of the two ends of the cross-linked double-stranded regions to form a cross-linking unit I.

In some more specific embodiments, the cross-linked double-stranded region has a length of 4bp or more, preferably 4 to 150bp, preferably 5 to 50bp, more preferably 6 to 30bp, and still more preferably 8 to 20 bp.

In some embodiments, the cross-linked core is a polypeptide formed by two or more amino acids linked to each other by peptide bonds, and may encompass dipeptides, tripeptides, tetrapeptides, oligopeptides, proteins, and the like.

In some embodiments, the crosslinked core is a nanoparticle, which is a microscopic particle on the order of nanometers, generally referring to a particle that is less than 100nm in at least one dimension. In particular, semiconductor nanoparticles smaller than 10nm are also referred to as quantum dots due to their electronic energy level quantization. Nanoparticles as cross-linked core encompass quantum dots, Fe₂O₃、Si、SiO₂Au and Ag nanoparticles, and the like.

In some embodiments, the crosslinked core is a polymeric compound, which is a compound having a relative molecular mass of several thousands to several millions, by connecting simple structural units in a repetitive manner. Specifically, the high molecular compound as the crosslinking core encompasses polylactic acid, polylactic acid-glycolic acid copolymer, polyethylene glycol, and the like.

For the second nucleic acid strand, one end of the second nucleic acid strand is attached to the crosslinking core, and the end thereof remote from the crosslinking core is a cohesive 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 mutual crosslinking of the crosslinking unit I and the scaffold unit is achieved by means of base complementary pairing.

In some embodiments, the second nucleic acid strand is a single-stranded nucleic acid strand (ssDNA), the entirety of the second nucleic acid strand acting as a cohesive end, effecting cross-linking with the scaffold unit. In some embodiments, the second nucleic acid strand may also be a double-stranded nucleic acid strand, and the end of the double-stranded second nucleic acid strand distal to the cross-linked core extends beyond ssDNA as a sticky end. In some embodiments, any of the second nucleic acid strands has the same nucleic acid sequence and its cohesive end is complementary to the sequence of the cohesive end of the first nucleic acid strand in the scaffold unit. In some embodiments, the cohesive ends of any second nucleic acid strand have the same nucleic acid sequence and are complementary to the sequence of the cohesive ends of the first nucleic acid strand in the scaffold unit. In some alternative embodiments, the sequence of the second nucleic acid strand, or the sequence of the sticky end of the second nucleic acid strand, may also be chosen from more than two, as long as the sequence of the sticky end of the first nucleic acid strand in the corresponding scaffold unit is capable of achieving complementarity thereto.

In some embodiments, the cohesive end of the second nucleic acid strand has a length of 4nt or more, which facilitates its stable crosslinking state under physiological conditions. Preferably, the length of the cohesive end of the second nucleic acid strand is 150nt or less, preferably 50nt or less, more preferably 30nt or less, more preferably 20nt or less. Specifically, the length of the cohesive end of the second nucleic acid strand is preferably 4 to 30nt, more preferably 4 to 20 nt. On the other hand, the length of the cohesive end of the second nucleic acid strand also affects the mechanical strength of the nucleic acid hydrogel finally obtained by crosslinking, and the mechanical strength of the nucleic acid hydrogel can be improved while achieving self-assembly of the nucleic acid hydrogel by designing the length of the cohesive end to be in the range of 4 to 20 nt.

< crosslinking Unit II >

The crosslinking unit II comprises at least 2 repeating segments comprising a single-stranded third nucleic acid strand and a double-stranded fourth nucleic acid strand, the crosslinking unit II is formed by alternating connection of the third nucleic acid strand and the fourth nucleic acid strand, and the third nucleic acid strand is complementary to a sequence of a cohesive end of the first nucleic acid strand. Specifically, each of the repeated segments comprises 1 single-stranded third nucleic acid strand and 1 double-stranded fourth nucleic acid strand, the third nucleic acid strand and the fourth nucleic acid strand being alternately linked to give a crosslinking unit II formed by alternately linking at least 2 third nucleic acid strands and at least 2 fourth nucleic acid strands. Illustratively, the number of 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., and the number of fourth 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 third nucleic acid strands and the number of the fourth nucleic acid strands in the present disclosure are not exhaustive, and may be any number suitable for cooperatively connecting the nucleic acid hydrogel structures of the scaffold unit and the crosslinking unit I to each other, so that the nucleic acid hydrogel has improved mechanical properties and can maintain the dynamic properties of the supramolecular hydrogel.

In some embodiments, the number of the third nucleic acid strands is at least 100 and the number of the fourth nucleic acid strands is at least 100. By adjusting the number of the third nucleic acid chain and the corresponding fourth nucleic acid chain, the number of the crosslinking sites provided by the crosslinking unit II can be adjusted, and further the network structure and the structural stability of the nucleic acid hydrogel can be adjusted. Further, in the cross-linking unit II, the third nucleic acid strand is a single-stranded nucleic acid strand, and the fourth nucleic acid strand is a double-stranded nucleic acid strand, that is, the cross-linking unit II is a long-chain cross-linking unit formed by alternately connecting a double-stranded region and a single-stranded region. Wherein the sequence of the third nucleic acid strand is complementary to the sequence of the cohesive end of the first nucleic acid strand in the scaffold unit, providing a crosslinking site for the crosslinking unit II and the scaffold unit. Through the molecular design mode, the scaffold units can be cooperatively connected, and self-assembly in the sequence in the cross-linking unit II can be avoided, so that the structural stability of the nucleic acid hydrogel is further improved, the sliding among nucleic acid chains is effectively inhibited, and the nucleic acid hydrogel with mechanical strength and dynamic characteristics of the supramolecular hydrogel is obtained.

In some preferred embodiments, the cross-linking unit II is an extended long-chain nucleic acid strand. The malleable structure of the cross-linking unit II is more suitable for being cross-linked with the single strand of the bracket, so that the cooperative connection of a hydrogel structure formed by cross-linking the bracket unit and the cross-linking unit I is realized, the sliding between the nucleic acid chains is better inhibited, and the instability caused by the gap of the complementary cohesive end is compensated.

In the present disclosure, the third nucleic acid strand or the fourth nucleic acid strand may be formed by polymerization of one or both of deoxyribonucleotides and ribonucleotides, respectively. And, as to any one of the nucleotides constituting the third nucleic acid strand or the fourth nucleic acid strand, it may be a modified or an unmodified nucleotide.

In the present disclosure, the nucleic acid structure of the third nucleic acid strand or the fourth nucleic acid strand may assume different spatial conformations. Illustratively, the nucleic acid may be an L-nucleic acid or a D-nucleic acid.

In some embodiments, the third nucleic acid strand is a single-stranded DNA formed from deoxyribonucleotides; in some embodiments, the fourth nucleic acid strand is a double-stranded DNA formed from deoxyribonucleotides.

In some embodiments, the length of the third nucleic acid strand is 4nt or more, preferably, the length of the third nucleic acid strand is 150nt or less, preferably 50nt or less, more preferably 30nt or less, more preferably 20nt or less. Specifically, the length of the third nucleic acid strand is preferably 4 to 30nt, more preferably 4 to 20 nt. In some embodiments, the length of the fourth nucleic acid strand is 4nt or more, preferably 10 to 40nt, more preferably 20 to 30 nt.

In some embodiments, the repeating segments are present in an amount of 0.5 to 99% based on the total moles of repeating segments in the crosslinking units I and II; optionally, the content of the repetitive fragment is 1-20%; preferably, the content of the repetitive fragment is 2.5-10%. Illustratively, the content of the repeating segments is 2.5%, 5%, 7.5%, 10%, 12%, 15%, 17%, 20%, and the like. The regulation and control of the mechanical modulus of the nucleic acid hydrogel can be controllably realized by regulating the adding proportion of the repeated segments. When the content of the repeating segment is 2.5 to 10%, the storage modulus G' of the nucleic acid hydrogel is increased and the mechanical strength is enhanced as the content of the repeating segment is increased.

Second aspect of the invention

A second aspect of the present disclosure provides the method for preparing a nucleic acid hydrogel according to the first aspect of the present disclosure, comprising the step of crosslinking and molding a scaffold unit with a crosslinking unit I and a crosslinking unit II.

In some embodiments, the method of preparing a nucleic acid hydrogel comprises the steps of:

preparing a scaffold unit in a gel matrix to obtain a scaffold unit solution;

< preparation of Stent Unit >

In some embodiments, the scaffold core in the scaffold unit is a nucleic acid, and the scaffold unit can be prepared by complementary pairing of nucleic acid fragments. Illustratively, the scaffold unit is a nucleic acid scaffold core to which three first nucleic acid strands are bound. Corresponding to this structure, three short-chain nucleic acid fragments were involved and designated Y1, Y2 and Y3.

The nucleic acid fragment Y1 includes a single-stranded region Y11, a single-stranded region Y12 and a single-stranded region Y13 which are sequentially linked, the nucleic acid fragment Y2 includes a single-stranded region Y21, a single-stranded region Y22 and a single-stranded region Y23 which are sequentially linked, and the nucleic acid fragment Y3 includes a single-stranded region Y31, a single-stranded region Y32 and a single-stranded region Y33 which are sequentially linked.

Wherein the sequence of the single-stranded region Y12 and the sequence of the single-stranded region Y33 are complementary sequences, the sequence of the single-stranded region Y13 and the sequence of the single-stranded region Y22 are complementary sequences, and the sequence of the single-stranded region Y23 and the sequence of the single-stranded region Y32 are complementary sequences.

The nucleic acid fragment Y1, the nucleic acid fragment Y2 and the nucleic acid fragment Y3 were placed in the same reaction system and annealed to obtain a scaffold unit having three first nucleic acid strands. In the scaffold unit, the single-stranded region Y12 of the nucleic acid fragment Y1 and the single-stranded region Y33 of the nucleic acid fragment Y3 are complementarily paired to form a scaffold double-stranded region, the single-stranded region Y13 of the nucleic acid fragment Y1 and the single-stranded region Y22 of the nucleic acid fragment Y2 are complementarily paired to form a scaffold double-stranded region, and the single-stranded region Y23 of the nucleic acid fragment Y2 and the single-stranded region Y32 of the nucleic acid fragment Y3 are complementarily paired to form a scaffold double-stranded region, which together constitute a scaffold core of the scaffold unit. And the single-stranded region Y11, the single-stranded region Y21 and the single-stranded region Y31 serve as three first nucleic acid strands attached to the scaffold core, respectively.

For a gel matrix, an aqueous medium may be used. The aqueous medium may be water or a buffer solution using water as a solvent. In some specific embodiments, the gel matrix is a phosphate buffer comprising 10mM phosphate, 100mM NaCl, pH 7.4. In addition, the gel matrix may be an aqueous medium under any other conditions capable of forming a nucleic acid hydrogel, and the present disclosure is not particularly limited thereto.

For the annealing treatment, the nucleic acid fragment Y1, the nucleic acid fragment Y2 and the nucleic acid fragment Y3 are incubated and then cooled until the nucleic acid fragment Y1, the nucleic acid fragment Y2 and the nucleic acid fragment Y3 are complementarily paired to form a scaffold unit with a double-stranded region. The incubation temperature, the incubation time and the cooling rate are determined by the specific sequence structures of the nucleic acid fragment Y1, the nucleic acid fragment Y2 and the nucleic acid fragment Y3, and the three can form the scaffold unit in a hybridization pairing mode.

In some embodiments, the scaffold core is a polypeptide and the scaffold unit is prepared by covalently binding the first nucleic acid strand to the polypeptide as a scaffold core. Preferably the scaffold unit is prepared by a click reaction, more preferably by a copper catalysed click reaction.

In some embodiments, wherein the scaffold core is a nanoparticle, the scaffold unit is prepared by covalently bonding the first nucleic acid strand to the nanoparticle as the scaffold core.

< preparation of crosslinking Unit I >

In some embodiments, the crosslinking core in crosslinking unit I is a nucleic acid, and the crosslinking unit can be prepared by complementary pairing of nucleic acid fragments. Illustratively, the crosslinking unit is obtained by binding 2 nucleic acid strands to a nucleic acid as a crosslinking core. Corresponding to this structure, 2 short-chain nucleic acid fragments were involved and designated L1 and L2.

The nucleic acid fragment L1 comprises a single-stranded region L11 and a single-stranded region L12 which are sequentially connected, and the nucleic acid fragment L2 comprises a single-stranded region L21 and a single-stranded region L22 which are sequentially connected. Wherein the sequence of single stranded region L12 and the sequence of single stranded region L22 are complementary sequences, and the sequence of single stranded region L11 is complementary to at least one of single stranded region Y11, single stranded region Y21 and single stranded region Y31 in the scaffold unit, and the sequence of single stranded region L12 is complementary to at least one of single stranded region Y11, single stranded region Y21 and single stranded region Y31 in the scaffold unit. Mutual crosslinking of the scaffold unit and the crosslinking unit I can be achieved by complementary pairing of the sequences of the single-stranded region L11, the single-stranded region L12, the single-stranded region Y11, the single-stranded region Y21 and the single-stranded region Y31.

In some embodiments, the sequence of single stranded region L11 is identical to the sequence of single stranded region L12, the sequence of single stranded region Y11, single stranded region Y21 and single stranded region Y31 are identical, and the sequence of single stranded regions L11, L12 is complementary to the sequence of single stranded regions Y11, Y12, Y13.

The nucleic acid fragment L1 and the nucleic acid fragment L2 were placed in the same reaction system and annealed to obtain a crosslinking unit I having two second nucleic acid strands. In the crosslinking unit I, the single-stranded region L22 of the nucleic acid fragment L1 complementarily paired with the single-stranded region L22 of the nucleic acid fragment L2 was a double-stranded region, and the single-stranded region L11 and the single-stranded region L21 became outwardly-extending cohesive ends of the double-stranded region away from the joining end, with the single-stranded region L11 and the single-stranded region L21 as 2 second nucleic acid strands joined to the crosslinking core, respectively.

In some more specific embodiments, cleavage sites are provided in single-stranded region L12 and single-stranded region L22, respectively, to allow cleavage of crosslinking unit I by the respective enzymes, thereby achieving a nucleic acid hydrogel-specific enzymatic response.

For the annealing treatment, after the nucleic acid fragment L1 and the nucleic acid fragment L2 were incubated, the temperature was decreased until the nucleic acid fragment L1 and the nucleic acid fragment L2 complementarily paired to form a scaffold unit having a double-stranded region. The incubation temperature, incubation time, and cooling rate are determined by the specific sequence structures of the nucleic acid fragment L1 and the nucleic acid fragment L2, and the two may be hybridized and paired to form the crosslinking unit I.

In some embodiments, the cross-linked core is a polypeptide, and the preparation of the cross-linking unit I is performed by covalently bonding the second nucleic acid strand to the polypeptide as the cross-linked core. Preferably the cross-linking units are prepared by a click reaction, more preferably by a copper catalysed click reaction.

In some embodiments, wherein the crosslinked core is a nanoparticle, the preparation of the crosslinking unit is performed by covalently bonding the second nucleic acid strand to the nanoparticle as the crosslinked core.

< crosslinking Unit II >

In some embodiments, the step of preparing the crosslinking unit II comprises:

mixing the long-chain single-stranded nucleic acid chain with a complementary chain in a gel matrix, and annealing, wherein the complementary chain is complementary to the sequence of the single-stranded region N1 to form a double-stranded fourth nucleic acid chain, and the single-stranded region N2 forms a third nucleic acid chain, so that a cross-linking unit II which is alternately connected with the fourth nucleic acid chain and the third nucleic acid chain is obtained; wherein, the adjacent 1 single-stranded third nucleic acid strand and 1 double-stranded fourth nucleic acid strand are connected to form 1 repeated segment, and the cross-linking unit II at least comprises 2 repeated segments.

For the long-chain single-stranded nucleic acid strand, a method in the art for realizing the preparation of a long-chain nucleic acid may be employed as long as a long-chain single-stranded nucleic acid strand formed by alternately connecting single-stranded regions N1 and N2 can be obtained. In some embodiments, the long-chain single-stranded nucleic acid strand is obtained by rolling circle amplification using a circular nucleic acid strand as a template. In some embodiments, the long-chain single-stranded nucleic acid strand is obtained by chemical synthesis.

In some embodiments, long-chain single-stranded nucleic acid strands are prepared in the presence of pyrophosphatase. Specifically, inorganic pyrophosphatase is added into the reaction system of rolling circle amplification for decomposing side product pyrophosphate to avoid forming DNA/MgP₂O₇A nanostructure. Further avoiding the agglomeration of the cross-linking unit II prepared by the long-chain single-chain nucleic acid chain, and spreading the structure of the cross-linking unit II as much as possible, which is beneficial to the serial connection of the bracket units.

For a circular nucleic acid strand, it can be obtained by connecting the linear template strands end to end. In some embodiments, the linear template strand comprises 2 single-stranded regions M1 and 2 single-stranded regions M2, the single-stranded regions M1 and M2 are alternately ligated to form a linear template strand, and the linear template strand is subjected to a circularization process to be ligated into a circular nucleic acid strand.

For rolling circle amplification, a circular nucleic acid chain with a single-stranded region M1 and a single-stranded region M2 alternately arranged is used as a template, a rolling change amplification primer is added, and after amplification reaction, a large number of single-stranded nucleic acid chains with a long chain formed by alternately connecting a single-stranded region N1 and a single-stranded region N2 are obtained. Wherein the sequence of the single-stranded region N1 and the sequence of the single-stranded region M1 are complementary sequences, and the sequence of the single-stranded region N2 and the sequence of the single-stranded region M2 are complementary sequences. And the sequence of the single-stranded region N2 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.

In some embodiments, the sequence of single stranded region N2 is identical to the sequence of single stranded region L11 in cross-linking unit I; in some embodiments, the sequence of single stranded region N2 is identical to the sequence of single stranded region L21 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 L11, single-stranded region L21; and the sequences of the single-stranded region Y11, the single-stranded region Y21 and the single-stranded region Y31 in the scaffold unit are identical, and the sequences of the single-stranded regions N2, L11 and L21 are complementary to the sequences of the single-stranded regions Y11, Y21 and Y31.

For the annealing treatment, a long-chain single-stranded nucleic acid strand is incubated with a complementary strand, and then the temperature is reduced until the complementary strand and the sequence of the single-stranded region N1 are complementarily paired to form a double-stranded fourth nucleic acid strand, and the single-stranded region N2 corresponds to form a single-stranded third nucleic acid strand. The incubation temperature, incubation time and cooling rate are determined by the specific sequence structures of the single-stranded region N1 and the complementary strand, so long as the two can form the fourth nucleic acid strand by hybridization pairing.

< scaffold Unit, crosslinking Unit I, crosslinking Unit II self-Assembly >

And mixing the support unit solution with the crosslinking unit solution, and self-assembling the support unit with the crosslinking unit I and the crosslinking unit II respectively to obtain the nucleic acid hydrogel.

In some embodiments, the scaffold unit solution is a solution of scaffold units comprising scaffold units obtained by dissolving nucleic acid fragments capable of assembling to form scaffold units in a gel matrix and then annealing the solution.

In some embodiments, the crosslinking unit solution is a crosslinking unit solution containing crosslinking unit I and crosslinking unit II obtained by dissolving a long-chain single-stranded nucleic acid strand, a complementary strand, and nucleic acid fragments capable of assembling to form crosslinking unit I in a gel matrix and annealing the solution.

In some embodiments, the solution of scaffold units is mixed with the solution of crosslinking units at a temperature of 4-50 deg.C, preferably 5-40 deg.C, more preferably 10-30 deg.C, such that the scaffold units base-complementarily pair with crosslinking units I and II, respectively, to achieve self-assembly of the nucleic acid hydrogel.

In some embodiments, the scaffold unit solution is mixed with the cross-linking unit solution at a pH of 3 to 11, preferably pH 4 to 10, more preferably pH 5 to 9, still more preferably pH 6 to 8.

In some embodiments, the molar ratio of the scaffold units in the scaffold unit solution to the total crosslinking units in the crosslinking unit solution is from 2:1 to 1:3, preferably from 1:1 to 1:2, more preferably 1: 1.

The nucleic acid hydrogel prepared by the method disclosed by the disclosure can be rapidly formed, has the advantages of simple preparation steps, easily realized conditions and the like, and can be rapidly prepared.

Third aspect of the invention

A third aspect of the present disclosure provides a 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 (a) to (c) below:

(a) as or in the preparation of biomedical materials;

(b) as or in the preparation of flexible electronic materials;

(c) as or to prepare a three-dimensional printed material.

The nucleic acid hydrogel has high mechanical strength and good stability, and has the dynamic characteristics of the supramolecular hydrogel, so that the nucleic acid hydrogel is suitable for being used as a biomedical material in the fields of drug delivery, cell culture differentiation, protein production, immune regulation and the like, or used as a flexible electronic material in the fields of wearable equipment, artificial skin, soft robots and the like, or used as a three-dimensional printing material in the field of 3D printing.

Examples

Embodiments of the present disclosure will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The experimental techniques and experimental procedures used in this example are, unless otherwise specified, conventional techniques, e.g., those in the following examples, in which specific conditions are not specified, and generally according to conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The materials, reagents and the like used in the examples are commercially available from normal sources unless otherwise specified.

Experimental Material

The information of the experimental reagents used in the following examples is shown in Table 1, and the designed DNA sequences are shown in Table 2 and the sequence listing.

TABLE 1

TABLE 2

Examples DNA sequences (including Y1, Y2, Y3, L1, L2, rolling circle amplification primers and the complementary strand of long-chain single-stranded DNA) were synthesized by a standard solid-phase phosphoramidite DNA synthesis method (Mermade-12DNA synthesizer, Bio Automation, USA) and isolated and purified by reverse-phase high performance liquid chromatography (Agilent 1200, Agilent, USA), and the purity of the starting material was characterized by LC-MS (Shimadzu 2020, Japan). The linear template strand in the examples was modified at the 5' end with a phosphate group and was purchased from Hippocampus Biotechnology Ltd, Beijing, China and purified by HPLC. All the water used for the experiments was ultrapure water produced by 18.2 M.OMEGA.cm Millipore. Other chemical reagents are superior pure and above.

Example 1 Preparation of nucleic acid hydrogels

Step 1, preparing long-chain single-stranded DNA by using rolling circle amplification reaction, which comprises the following steps:

350pmol of the linear template strand, 10. mu.L of 10 XSSDA/RNA ligase reaction buffer, 3. mu.L of ssDNA/RNA ligase, and 5. mu.L of 50mM MnCl₂Mixing well, adding appropriate amount of high purity water to total volume of 100 μ L. And (3) incubating at 60 ℃ for 6h for cyclization reaction, reacting at 80 ℃ for 10min to inactivate ligase, and standing at room temperature for 10min to cool to room temperature to obtain circular template DNA.

To the above system was added 5. mu.L of exonuclease I and 2.5. mu.L of exonuclease III. The acyclic linear template strand was digested at 37 ℃ for 30min, then inactivated at 90 ℃ for 5min, and cooled at room temperature for 10 min. The reaction product was subjected to denaturing gel electrophoresis using 20% acrylamide/8M urea, and the cyclization or ligation products were observed. The purification was carried out by ultrafiltration 3 times using an ultrafiltration tube with a molecular weight cut-off of 3000 Da.

The components shown in Table 3 were mixed and incubated at 30 ℃ for 4h, followed by incubation at 65 ℃ for 10min to inactivate the enzyme. Inorganic pyrophosphatase is added into the reaction system for decomposing by-product pyrophosphate to avoid forming DNA/MgP₂O₇Nanostructure, resulting in free long-chain single-stranded DNA.

TABLE 3

Components	Volume (μ l)	Final concentration of 100. mu.l system
			Phi29 DNA polymerase reaction buffer	10	1×
BSA	1	200μg/mL
			dNTPs	10	1mM
Endless template chain	60	500nM
			Rolling circle amplification primer	1.5	1μM
phi29DNA polymerase	5	0.2U/μl
			Inorganic pyrophosphatase	5	0.005U/μl
Ultrapure water	2.5

The absorbance of the long-chain single-stranded DNA at 260nm was measured using an ultraviolet spectrophotometer (Nanodrop 2000, Thermo Scientific). The extinction coefficient of the repeat sequence at 260nm was used to calculate the amount of all repeats in the long-stranded single-stranded DNA.

And 2, mixing 20nmol of Y1, Y2 and Y3 respectively, putting the aqueous solution into a centrifugal tube, and freeze-drying. To the lyophilized product, 20. mu.l of phosphate buffer (PBS, 10mM phosphate, 100mM NaCl, pH 7.4) was added, heated at 95 ℃ for 5min, and naturally cooled to room temperature to obtain a scaffold unit solution.

And 3, mixing 29.25nmol of L1 and L2, the complementary strand of the long-chain single-stranded DNA containing 1.5nmol of repetitive fragments and the complementary strand of the 1.5nmol of long-chain single-stranded DNA, wherein the amount of the complementary strand is equal to that of the long-chain single-stranded DNA repetitive sequences, and the total amount of the L1, the L2 and the long-chain single-stranded DNA repetitive sequences is 60 nmol. Freeze drying, adding 20 μ l phosphate buffer, heating at 95 deg.C for 5min, and naturally cooling to room temperature to obtain crosslinking unit solution with repeating segment molar content of 2.5% in crosslinking unit II.

And 4, mixing the scaffold unit solution and the crosslinking unit solution at room temperature, and oscillating and centrifuging to obtain the DNA supramolecular hydrogel with different crosslinking unit II contents, namely the nucleic acid hydrogel.

Example 2 Preparation of nucleic acid hydrogels

The nucleic acid hydrogel was prepared by the method shown in step 1 to step 4 of example 1, wherein the solution of the crosslinking unit in step 3 was prepared as follows:

28.50nmol of each of L1 and L2, a long-chain single-stranded DNA solution containing 3.0nmol of the repeat sequence, and 3.0nmol of the complementary strand were mixed and freeze-dried to obtain a crosslinking unit solution containing 5.0% by mole of the repeat fragment in the crosslinking unit II.

Example 3 Preparation of nucleic acid hydrogels

27.75nmol each of L1 and L2, a long-chain single-stranded DNA solution containing 4.5nmol of the repeat sequence, and 4.5nmol of the complementary strand were mixed and freeze-dried to obtain a crosslinking unit solution having a repeating fragment molar content of 7.5% in the crosslinking unit II.

Example 4 Preparation of nucleic acid hydrogels

27.00nmol each of L1 and L2, a solution of a long-chain single-stranded DNA containing 6.0nmol of the repeat sequence, and 6.0nmol of the complementary strand were mixed and freeze-dried to obtain a solution of a crosslinking unit having a repeating fragment content of 10.0% by mole in the crosslinking unit II.

Comparative example 1

The nucleic acid hydrogel was prepared by the method shown in step 2 to step 4 of example 1, wherein the solution of the crosslinking unit in step 3 was prepared as follows:

30nmol each of L1 and L2 was mixed and freeze-dried to obtain a crosslinking unit solution containing no crosslinking unit II.

Performance testing

The rheological properties of each hydrogel sample were tested using a Kinexus Pro + rotational rheometer manufactured by malvern, england. A parallel plate rotor with the diameter of 8mm is selected for testing, and the distance between the test bench and the parallel plate is set to be 150 mu m. And (3) placing a 40 mu L hydrogel sample in the center of a test bench, slightly scraping redundant gel around by using a medicine spoon after a rotor is pressed down, and dropwise adding a small amount of silicone oil around to seal so as to prevent water from volatilizing.

The nucleic acid hydrogels of examples 1 to 4 and comparative example 1 were tested for storage modulus G' and loss modulus G "at a fixation temperature of 25 ℃, a strain amplitude of 1%, and an oscillation frequency of 1 Hz. Examples 1-4 and comparative example 1 each tested three replicates and the statistical results are shown in figure 2. The storage modulus G' reflects the elasticity, i.e. the rigidity, of the material, the greater the storage modulus, the less easily the material is deformed. As can be seen from FIG. 2, the storage modulus G 'of the nucleic acid hydrogels of each example was larger than that of the hydrogel of the comparative example, and the G' of the nucleic acid hydrogels gradually increased as the content of the repeating segment in the crosslinking unit II increased. Thus showing that the mechanical property of the nucleic acid hydrogel is effectively improved.

Due to the reversibility and dynamic property of the supermolecule interaction, the nucleic acid hydrogel with enhanced mechanics has the self-repairing characteristic. Two separately prepared hydrogels of example 2 adhered together after bonding and the two hydrogels gradually fused after standing at room temperature for a period of time without a distinct interface. The rheological properties of the hydrogel before and after self-healing were measured using a rheometer, and the mechanical modulus at 25 ℃, 1% strain amplitude, and 1Hz oscillation frequency was as shown in fig. 3, with no significant difference between the mechanical modulus of the hydrogel before and after self-healing.

The mechanical enhancement of DNA hydrogel can retain the shear thinning and injectable properties of supermolecular hydrogel. As shown in FIG. 4, the hydrogel of example 2 was in a gel state in the syringe before injection, and the viscosity decreased by the shearing force of the inner wall of the needle during injection, and was able to smoothly pass through the needle. After the injection is finished, the sample is quickly recovered to a gel state to form a fibrous gel filament.

The above examples of the present disclosure are merely examples provided for clearly illustrating the present disclosure and are not intended to limit the embodiments of the present disclosure. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present disclosure should be included in the protection scope of the claims of the present disclosure.

Sequence listing

<110> Qinghua university

<120> nucleic acid hydrogel with improved mechanical properties, and preparation method and application thereof

<130> 6044-2123479I

<141> 2021-04-13

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Claims

1. A nucleic acid hydrogel, wherein the nucleic acid hydrogel comprises a scaffold unit, and a crosslinking unit I and a crosslinking unit II which are respectively crosslinked with the scaffold unit;

2. The nucleic acid hydrogel according to claim 1, wherein the content of the repeating segments is 0.5-99% based on the total number of moles of the repeating segments in the crosslinking unit I and the crosslinking unit II; optionally, the content of the repetitive fragment is 1-20%; preferably, the content of the repetitive fragment is 2.5-10%.

3. The nucleic acid hydrogel of claim 1 or 2, wherein the materials forming the scaffold core or the cross-linked core are independently from each other selected from the group consisting of: nucleic acids, polypeptides, polymeric compounds, and nanoparticles.

4. The nucleic acid hydrogel according to any one of claims 1 to 3, wherein any nucleotide of any one of the first, second, third and fourth nucleic acid strands is a modified nucleotide or an unmodified nucleotide;

5. The nucleic acid hydrogel according to any one of claims 1 to 4, wherein the length of the cohesive end of any one of the first nucleic acid strand and the second nucleic acid strand is 4nt or more, preferably 4 to 30nt, more preferably 4 to 20 nt;

6. The nucleic acid hydrogel according to any one of claims 1 to 5, wherein the length of the fourth nucleic acid strand is 4nt or more, preferably 10 to 40nt, more preferably 20 to 30 nt.

7. A method for producing the nucleic acid hydrogel according to any one of claims 1 to 6, wherein the method comprises a step of crosslinking a scaffold unit with a crosslinking unit I and a crosslinking unit II to shape.

8. The method according to claim 7, wherein the method comprises the steps of:

preparing a scaffold unit in a gel matrix to obtain a scaffold unit solution;

9. The method of claim 8, wherein the step of preparing the crosslinking unit II comprises:

10. A method of preparing a nucleic acid hydrogel, comprising the steps of:

wherein the end of the first nucleic acid strand distal to the scaffold core is a sticky end and is complementary to the sequence of the second nucleic acid strand distal to the sticky end of the cross-linking core; said third nucleic acid strand being complementary to the sequence of the cohesive end of said first nucleic acid strand;

11. Use of the nucleic acid hydrogel of any one of claims 1 to 6, or the nucleic acid hydrogel prepared by the method of any one of claims 7 to 10, in at least one of the following (a) to (c):

(a) as or in the preparation of biomedical materials;

(b) as or in the preparation of flexible electronic materials;

(c) as or to prepare a three-dimensional printed material.