DNA nanostructures as antifreeze agents

Abstract

The present invention discloses the use of a DNA nanostructure as an antifreeze agent. DNA, a biomacromolecule essential for the development and normal functioning of organisms, possesses excellent programmability and biocompatibility. This invention provides a general approach to the antifreeze field, using specific types of DNA nanostructures as antifreeze agents. The hydrophobic bases of DNA tend to adsorb onto tiny ice crystals, stabilizing the entire material system with the help of hydrophilic ribose phosphate chains. This Kelvin effect achieves excellent ice suppression, effectively inhibiting ice recrystallization at relatively low concentrations. The DNA nanostructures used as antifreeze agents in the present invention have simple and rapid preparation processes, good biocompatibility, and structural predictability. Different material sizes and morphologies can be designed to achieve varying antifreeze effects.

Description

Technical Field

[0001] The invention belongs to the technical field of antifreeze bionic nanomaterials, and particularly relates to use of a DNA nanostructure as an antifreeze agent. Background Art

[0002] Recrystallization is the primary cause of cell damage during cryopreservation. Currently, the most commonly used cryopreservation method involves adding high concentrations of permeating cryoprotectants, such as dimethyl sulfoxide (DMSO) or organic solvents like glycerol, to cells to minimize ice formation. However, high concentrations of organic solvents are often biotoxic, and cell survival rates after cryopreservation using this method are often unsatisfactory, presenting a significant challenge in cell cryopreservation.

[0003] Antifreeze proteins (AFPs) have been shown to be present in many polar organisms and exhibit thermal hysteresis and ice recrystallization inhibition (IRI) activity even at extremely low concentrations. However, extracting natural AFPs from organisms is a time-consuming and complex process. Furthermore, their dynamic ice formation and potential biotoxicity limit their large-scale application in antifreeze applications. While some AFPs have been artificially synthesized or expressed, these proteins are generally costly, have poor biocompatibility, and exhibit low thermal stability.

[0004] To overcome the above problems, some artificially synthesized AFPs biomimetic materials have been developed, including polymers, small molecules, and nanomaterials. For example, polyvinyl alcohol can inhibit the growth of ice crystals and improve the cell recovery rate after cryopreservation at concentrations below 0.1% wt; carbon nitride-like quantum dots and graphene oxide have shown significant activity in experiments on ice recrystallization inhibition activity. It has also been verified that the size and degree of crowding of polymers may increase the IRI activity of "inactive" polymers; in addition, low molecular weight carbohydrate surfactants can also inhibit ice recrystallization; and conjugates of gold colloids and peptides also have significant IRI activity. However, these artificially synthesized AFPs biomimetic materials still inevitably have problems such as potential biological toxicity and difficulty in synthesis. Therefore, the development of programmable nano-antifreeze agents with extremely significant IRI activity, good biocompatibility, and easy large-scale synthesis remains an urgent problem to be solved. Summary of the Invention

[0005] In light of this, the present invention provides the use of DNA nanostructures as antifreeze agents. The inventors surprisingly discovered that DNA, a biomacromolecule essential for the development and normal functioning of organisms, possesses excellent programmability and biocompatibility. Due to the hydrophobicity of DNA bases, DNA nanostructures tend to adsorb onto ice crystals, while the hydrophilic ribose phosphate chains help the nanomaterials remain stable in ice-water systems. DNA nanostructures can achieve excellent ice suppression due to the Kelvin effect, effectively inhibiting ice recrystallization at relatively low concentrations.

[0006] In order to achieve the above object, the scheme of the present invention is as follows:

[0007] The present invention first provides a use of a DNA nanostructure as an antifreeze agent. The DNA nanostructure is a nanostructure formed by self-assembly of a DNA sequence; or a spherical nucleic acid formed by coupling a DNA single strand with a terminal thiol group modified on the surface of a rigid core.

[0008] A further approach is to design DNA nanostructures of different sizes and morphologies based on the programmable properties of DNA, and obtain different antifreeze effects due to the differences in the density of ice binding sites.

[0009] In a further solution, the DNA nanostructure is directly added to achieve antifreeze.

[0010] In a further embodiment, the DNA nanostructure is a nanostructure formed by self-assembly of a DNA sequence;

[0011] Preferably, the DNA nanostructure is a spherical structure, which is self-assembled by A-containing bases;

[0012] Preferably, the DNA nanostructure is an irregular sheet-like structure, which is self-assembled by C-containing bases or G-containing bases.

[0013] In a further embodiment, the DNA nanostructure is used as an antifreeze agent at a concentration of 0.5-5 mg / mL.

[0014] In a further embodiment, the antifreeze is used for cell freezing, and 10 mg of antifreeze is added to 4×10 5 in a cell.

[0015] In a further embodiment, the DNA nanostructure is a spherical nucleic acid formed by coupling a single strand of DNA with a terminal thiol group to the surface of a rigid core;

[0016] Preferably, the particle size of the rigid core is 15-85 nm; more preferably 30-85 nm;

[0017] Preferably, the rigid core is a noble metal nanoparticle;

[0018] Preferably, the noble metal nanoparticles are gold nanoparticles.

[0019] In a further embodiment, the DNA nanostructure is used as an antifreeze agent, with the rigid core as a quantitative standard, and its added concentration is 0.1-10 nM.

[0020] In a further embodiment, the antifreeze agent is used for cell freezing, and the core concentration of spherical nucleic acid is used as a quantitative standard. Every 0.4 nM antifreeze agent is added to 4×10 5 in a cell.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides a general idea in the field of antifreeze, using certain specific types of DNA nanostructures as antifreeze agents. The hydrophobic bases of DNA tend to adsorb on tiny ice crystals and stabilize the entire material system with the help of hydrophilic ribose phosphate chains. Due to the Kelvin effect, a good ice-inhibiting effect is achieved, thereby effectively inhibiting the recrystallization of ice crystals at lower concentrations.

[0023] As antifreeze agents, DNA nanostructures have simple and rapid preparation processes, good biocompatibility, and programmable structure prediction. Different material sizes and morphologies can be designed according to needs to achieve different antifreeze effects.

[0024] Compared with known antifreeze materials, DNA nanostructures require lower concentrations and smaller amounts as antifreeze agents, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a flow chart for preparing an antifreeze agent containing a DNA nanostructure according to a typical embodiment of the present invention;

[0026] Figure 2 TEM characterization images of different DNA nanostructures obtained after self-assembly of DNA single strands with different programmed structures and ferrous ions in Example 1;

[0027] Figure 3 TEM characterization images of DNA nanostructures of different sizes obtained after self-assembly of DNA single strands of different base lengths with ferrous ions in Example 2;

[0028] Figure 4 TEM characterization images of spherical nucleic acid nanoantifreeze agents of different sizes obtained by assembling thiol-modified DNA single strands with GNPs of different sizes in Example 3;

[0029] Figure 5The optical photographs and size change diagrams of ice crystals of pure water and five self-assembled DNA nanoantifreeze agents after 0, 10, 20 and 30 minutes of ice crystal recrystallization in Example 4 are shown;

[0030] Figure 6 The optical photographs and size change diagrams of ice crystals of pure water and four spherical nucleic acid nano-antifreeze agents after 30 minutes of ice crystal recrystallization in Example 5 are shown;

[0031] Figure 7 This is an optical photograph of ice crystals of the spherical nucleic acid nanoantifreeze modified with GNPs of 85 nm in diameter and DNA of different chain lengths after recrystallization at a concentration of 0.4 nM for 30 minutes in Example 6;

[0032] Figure 8 These are the cell recovery rate and cell viability test results in Example 7. DETAILED DESCRIPTION

[0033] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] The present invention provides the use of DNA nanostructures as antifreeze agents. Due to the hydrophobicity of DNA bases, the DNA nanostructures tend to adsorb onto ice crystals. The hydrophilic ribose phosphate chains help the nanomaterials remain stable in the ice-water system. Due to the Kelvin effect, a good ice-inhibiting effect can be achieved, and the recrystallization of ice crystals can be effectively inhibited at lower concentrations.

[0036] The inventors used a programmable temperature-controlled cryogenic stage and an optical microscope to apply a specially structured DNA nanostructure as an antifreeze agent. The structure inhibits ice recrystallization and significantly reduces ice crystal size. The DNA nanostructure described herein can be added directly to achieve an antifreeze effect, making it simple to use and providing excellent antifreeze efficacy.

[0037] The DNA nanostructures used in this study for antifreeze applications fall into two categories: one is a nanostructure formed by self-assembly of DNA sequences; the other is a densely assembled DNA shell adsorbed onto the surface of a rigid core, specifically a spherical nucleic acid formed by coupling a single strand of DNA with a terminal thiol group to the surface of the rigid core. Because the length, type, and number of base sequences in these DNA nanostructures are controllable and programmable, DNA nanostructures of varying shapes and sizes can be designed to alter recrystallization inhibition activity. Furthermore, due to the programmable nature of DNA sequences, the type and length of bases can be varied to form DNA nanostructures of varying shapes and sizes, thereby varying the density of ice-binding sites on the DNA nanostructures, leading to differences in antifreeze activity and meeting diverse antifreeze requirements.

[0038] The following will provide a detailed description of the two types of DNA nanostructures mentioned in this article to make the scheme of using DNA nanostructures as antifreeze agents in this article clearer.

[0039] In some typical embodiments of the present invention, the DNA nanostructure used as an antifreeze agent is a nanostructure formed by self-assembly of DNA sequences. The process of self-assembly of DNA sequences into nanostructures can refer to the known technology in the art. The more common method is to drive DNA self-assembly through the strong coordination effect between ferrous ions and nitrogen atoms and oxygen atoms on DNA nucleobases. For example, in some specific embodiments of the present invention, the self-assembly of DNA sequences into DNA nanostructures can refer to Figure 1 As shown in , it mainly includes the following steps:

[0040] Providing a DNA sequence, wherein the DNA sequence is a single base sequence or a random sequence with a length of 5-20 bases;

[0041] The ferrous ions are mixed evenly with the DNA sequence. Specifically, the DNA sequence is prepared into an aqueous solution of a certain concentration, and then an aqueous solution containing ferrous ions is added to the solution. The strong coordination between the ferrous ions and the nitrogen and oxygen atoms on the DNA nucleobases drives the DNA to self-assemble. It should be noted that after the addition of the ferrous ions, annealing is required at the melting point of the DNA for 2-3 hours. Finally, the product is centrifuged, washed, freeze-dried, and resuspended in an aqueous phase for use as an antifreeze.

[0042] Preferably, during the self-assembly process of the DNA sequence, the molar ratio of ferrous ions to DNA sequence is between 2:1 and 20:1.

[0043] It is understandable that by changing the base type and sequence length, DNA nanostructures with different morphologies and sizes can be prepared, thereby achieving different antifreeze effects:

[0044] In some exemplary embodiments of the present invention, DNA nanostructures are self-assembled from "A"-containing bases. Depending on the proportion of "A" bases, the DNA nanostructures exhibit spherical structures ranging from 20 to 300 nm. Furthermore, as the proportion of "A" bases increases, the size of the resulting DNA nanostructures increases.

[0045] In other exemplary embodiments of the present invention, DNA nanostructures are self-assembled from C-containing or G-containing bases, exhibiting irregular sheet-like structures. Furthermore, as the proportion of C and G bases increases, the size of the resulting DNA nanostructure decreases.

[0046] Furthermore, when the DNA nanostructure is used as an antifreeze, its concentration is 0.5-5 mg / mL. In specific applications, when the DNA nanostructure is used as an antifreeze for cell freezing, preferably, every 10 mg of antifreeze is added to 4×10 5 in a cell.

[0047] This type of self-assembled DNA nanostructure has different antifreeze activities depending on the final structure. Specifically, the spherical DNA nanostructure forms point contact with the surface of the ice crystal, and the density of ice binding sites is lower. Therefore, at the same concentration, the inhibitory activity on ice crystal recrystallization is lower than that of the sheet-like DNA nanostructure that forms surface contact with the ice crystal surface.

[0048] In other typical embodiments of the present invention, the DNA nanostructure used in the antifreeze agent is a spherical nucleic acid formed by coupling a single strand of DNA with a terminal thiol group to the surface of a rigid core. Specifically, a single base sequence or a random sequence with a length of 5-20 bases is used as the DNA sequence, and the DNA sequence is coupled to the surface of the rigid core through terminal modification. Preferably, the rigid core is a noble metal nanoparticle, and more preferably, the noble metal nanoparticle is a gold nanoparticle. The spherical nucleic acid is formed by the terminal thiol group modification of the DNA sequence and the noble metal nanoparticle through the metal-thiol interaction. The specific synthesis route can be referred to Figure 1 The specific preparation method can also refer to the existing technology, so it will not be described in detail here.

[0049] This spherical nucleic acid can be used as an antifreeze agent, and its antifreeze activity can be adjusted by adjusting the length of the DNA single strand and the size of the rigid core. For example, within a certain range, as the length of the DNA single strand increases, its antifreeze activity also increases. However, due to steric hindrance, after the single strand length increases to a certain extent, further increase in single strand length makes it more difficult for the DNA to couple with the rigid core surface, and its antifreeze activity decreases. Therefore, the preferred length of the DNA single strand is between 5 and 20 bases.

[0050] In addition, as the size of the rigid core increases, more DNA single strands can be coupled to its surface to provide more ice-binding sites, thereby obtaining better recrystallization inhibition activity. The specific activity can be adjusted according to the actual antifreeze requirements. If the particle size is too small, the antifreeze effect will be reduced, and if the particle size is too large, it will be difficult to prepare. Therefore, preferably, the particle size of the rigid core is between 15-85 nm, more preferably between 30-85 nm.

[0051] Furthermore, when the spherical nucleic acid is used as a DNA nanostructure antifreeze agent, the rigid core is used as a quantitative standard, and the concentration of the DNA nanostructure is between 0.1-10 nM. It is understood that the concentration of the DNA nanostructure can vary, and the desired antifreeze effect can be achieved by adjusting the concentration of different DNA nanostructures according to the size of different rigid cores. In specific applications, the antifreeze agent can be directly added. When the DNA nanostructure is used as an antifreeze agent for cell freezing, the core concentration of the spherical nucleic acid is used as a quantitative standard, and every 0.4 nM antifreeze agent is added to 4×10 5 in a cell.

[0052] The DNA nanostructures described in this article exhibit significant recrystallization inhibition activity upon direct addition, enabling their direct use in cryopreservation to protect cells from ice crystal damage. This significant ice crystal recrystallization inhibition effect is evident even at relatively low concentrations. Furthermore, the DNA nanostructures are simple and rapid to prepare, exhibit excellent biocompatibility as cryoprotectants, and possess programmable structural predictions, enabling the design of diverse material morphologies and sizes to meet diverse cryoprotection needs.

[0053] The present invention is described below by means of specific examples. It should be noted that the following specific examples are for illustrative purposes only and do not limit the scope of the present invention in any way. In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used are all commercially available.

[0054] Example 1

[0055] DNAs with different base sequences (AAAAAAAAAA, abbreviated as A10; AATTTTTTTT, abbreviated as A2T8; AAAATTTTTT, abbreviated as A4T6; AAAAAATTTT, abbreviated as A6T4; AAAAAAAATT, abbreviated as A8T2; CCCCCCCCCC, abbreviated as C10; GGGGGGGGGG, abbreviated as G10) were prepared into 50 μM aqueous solutions respectively;

[0056] 30 μL of FeCl2·4H2O aqueous solution (20 mM) was added to each of them, and after rapid mixing by vortex oscillation, the mixture was placed at 95°C and annealed for 3 hours; centrifuged at 13000 rpm for 15 minutes, repeated centrifugation three times, and resuspended in pure water to prepare self-assembled DNA nano antifreeze agents with different concentrations (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 5 mg / mL).

[0057] The microscopic morphology of different base sequences and ferrous chloride complexes was observed using transmission electron microscopy. Figure 2 As shown in Figure 2, due to the different binding affinities of different nucleotides for metal ions, the self-assembled DNA nanostructure driven by ferrous ion coordination is programmable. Sequences containing "A" bases tend to form spherical nanoparticles. As the proportion of "A" bases increases, the size of the formed nanoparticles increases, with A10 reaching 220nm. C10 and G10 tend to assemble into nanosheets.

[0058] Example 2

[0059] DNA of different base lengths (AAAAAAAAAAAAAAAAAAAAA, referred to as A20; AAAAA, referred to as A5) were prepared into 50 μM aqueous solutions;

[0060] 30 μL of FeCl2·4H2O aqueous solution (20 mM) was added thereto respectively, and the mixture was quickly mixed by vortex oscillation. The mixture was placed at 95°C and annealed for 3 h; centrifuged at 13000 rpm for 15 min, repeated centrifugation 3 times, and then resuspended in pure water.

[0061] The microscopic morphology of different base sequences and ferrous chloride complexes was observed using transmission electron microscopy. Figure 3 Nucleic acids of different lengths coordinate with metal ions to form DNA nanostructures with similar morphologies but different sizes. At the same concentration, the sequence length is positively correlated with the size of the nanostructure.

[0062] Example 3

[0063] Preparation of gold nanoparticles (GNPs): Add 1 mL of 3% (v / v) C6H5Na3O7·2H2O to 98.9 mL of pure water, then quickly add 0.1 mL of 9.8% (v / v) HAuCl4·4H2O, keep boiling for about 7 minutes, and synthesize GNPs of different sizes (15 nm, 30 nm, 60 nm, 85 nm) by the seed growth method.

[0064] Preparation of spherical nucleic acids: Single-stranded DNA modified with thiol groups (SH-AAAAAAAAAA, abbreviated as SH-A10) and synthesized GNPs of different sizes were assembled into spherical nucleic acids by instantaneous dehydration with n-butanol. Specific steps: Take 100 μL of GNPs of a certain concentration and add the thiol-modified single-stranded DNA. Then add 20 μL of 5 mM tris(2-carboxyethyl)phosphine to break the disulfide bonds between the DNA. After mixing, add 900 μL of n-butanol and vortex dehydration. Then add 200 μL of 0.5× TBE buffer, vortex again, and centrifuge at 5000 rpm for 3 minutes. Remove the supernatant and wash with pure water three times. Resuspend in water to prepare DNA nanostructure antifreeze agents of different concentrations (0.1-10 nM).

[0065] Figure 4 Figure 3 shows the microscopic morphology of spherical nucleic acids assembled with SH-A10 and four different sizes of gold nanoparticles of 15, 30, 60 and 85 nm prepared in this example.

[0066] Example 4

[0067] The five self-assembled DNA nanostructures A10, A8T2, A4T6, C10 and G10 in Example 1 were prepared into a solution with a concentration of 3.0 mg / mL with pure water. 10 μL of the self-assembled DNA nanoantifreeze agent composed of the above single-stranded DNA and pure water were respectively dropped vertically from a height of 1.6 m onto a glass slide pre-cooled with liquid nitrogen, instantly forming circular ice crystals with a thickness of about 10 μm. The ice crystals were then quickly transferred to a sealed cold stage chamber at 60°C and heated to 8°C at a rate of 15°C / min. After maintaining the temperature for 30 minutes, photos were taken with an optical microscope. The average size of the 10 largest ice crystals was selected using Image J software and compared with that of pure water to observe the antifreeze activity of the self-assembled DNA nanostructures. The results are as follows: Figure 5 As shown in .

[0068] pass Figure 5 The results show that the optical photographs and size changes of ice crystals of pure water and five DNA nano-antifreeze agents after 30 minutes of recrystallization show that C10 and G10 tend to self-assemble into sheet-like nanostructures, forming a larger area of ​​contact with the surface of ice crystals, and therefore have better antifreeze effects than the spherical nanostructures formed by self-assembly of sequences containing "A" bases.

[0069] Example 5

[0070] The four spherical nucleic acid nanoparticles of different sizes in Example 3 were prepared into a solution with a concentration of 0.4 nM with pure water. 10 μL of each spherical nucleic acid nanomaterial was dropped vertically from a height of 1.6 m onto a glass slide pre-cooled with liquid nitrogen, instantly forming a circular ice sheet with a thickness of about 10 μm. The solution was then quickly transferred to a sealed cold stage chamber at 60°C, and the temperature was raised to 8°C at a rate of 15°C / min. The temperature was finally maintained for 30 minutes, and then photographs were taken with an optical microscope to observe the antifreeze activity of the spherical nucleic acid nano antifreeze agent. The results are as follows: Figure 6 As shown in .

[0071] pass Figure 6 The results in the figure show the changes in ice crystals of the four spherical nucleic acid nanomaterials after 30 minutes of ice crystal recrystallization. Since larger gold nanoparticles can couple with more DNA single strands, the antifreeze effect of spherical nucleic acid nano antifreeze agents increases with the increase of gold nanoparticle size.

[0072] Example 6

[0073] GNPs with a diameter of 85 nm were coupled to three thiol-modified DNA single chains of different lengths, SH-GGGGG, SH-GGGGGGGGGGGG, and SH-GGGGGGGGGGGGGGG (the specific preparation method is the same as in Example 3) to form three spherical nucleic acid nanoantifreeze agents, GNP85-G5, GNP85-G10, and GNP85-G14. Pure water was used to prepare solutions with a concentration of 0.4 nM. 10 μL of spherical nucleic acid nanomaterials of different chain lengths were respectively dropped vertically from a height of 1.6 m onto a glass slide pre-cooled with liquid nitrogen, instantly forming circular ice sheets with a thickness of about 10 μm. The solutions were then quickly transferred to a sealed cold stage chamber at 60°C, heated to 8°C at a rate of 15°C / min, and finally maintained for 30 min. Optical microscope photos were taken to observe the antifreeze activity of the spherical nucleic acid nanoantifreeze agents. The results are shown in FIG. Figure 7 As shown in .

[0074] pass Figure 7 It can be seen that all three spherical nucleic acids exhibited ice crystal recrystallization inhibition effects, among which GNP85-G10 had the best recrystallization inhibition effect. This is because within a certain range, the increase in the length of the DNA single chain leads to an increase in antifreeze activity; but due to the steric hindrance effect, further increasing the length of the single chain will make it more difficult for DNA to couple with the gold nanoparticle surface, so the antifreeze activity of GNP85-G14 has decreased to a certain extent.

[0075] Example 7

[0076] In this example, the antifreeze formed by the aforementioned DNA nanostructure was used to freeze the DU145 cells after amplification and culture.

[0077] Specifically, the C10 self-assembled sheet-like DNA nanostructures in Example 3 were dispersed into MEM culture medium to prepare 100 μL of culture medium mixture containing DNA nanostructures, and then 100 μL of DU145 cells (cell density of 4×10 5 / mL), and transferred to cryopreservation tubes and mixed evenly to make the final concentrations of C10 nanosheets reach 0, 0.1, 0.3, 0.6, 0.8, 1.0, and 1.1 mg / mL respectively;

[0078] 10% (V / V) DMSO was used as the control group, and equal volumes of MEM medium and cells were added;

[0079] After incubation for 10 min, each group was frozen to −80°C at 1°C / min, stored at this temperature for 24 h, and then rapidly thawed in a 37°C water bath and rinsed three times with PBS;

[0080] Annexin V-FITC / PI fluorescent dye was added to each group of cells after thawing, and the recovery rate of cells after freezing and cell viability at different times were determined by flow cytometry. The results are as follows: Figure 8 As shown in .

[0081] Figure 8 The cell recovery rate in the experiment proved that the cryopreservation effect of C10 nanosheets at a concentration of 1.0 mg / mL on cells was comparable to that of the commonly used cryopreservative 10% (V / V) DMSO, and the frozen cells still had strong viability after 24 hours, demonstrating that the DNA nanostructures of the present invention can be directly used as antifreeze agents and have a good ability to protect cells from low-temperature damage during the cryopreservation process.

[0082] Through the above examples, it can be seen that at micromolar concentrations (1μM-1000μM), the ice crystal size of the experimental group adding DNA self-assembled nano antifreeze can reach 50-70μm, while the ice crystal size of the experimental group adding spherical nucleic acid nano antifreeze can reach 20-50μm. Taking pure water as the control group, the inhibitory effect of DNA nano antifreeze on ice crystal recrystallization can be reduced to 15-50% of the control group. This shows that DNA nanostructures have excellent antifreeze effects. It can be seen from the cell freezing experiments conducted simultaneously that when DNA nanostructures are used as antifreeze agents, they not only have excellent effects and ensure the viability of cells, but also can well protect cells from low-temperature damage during freezing.

[0083] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0084] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. Use of DNA nanostructures as ice recrystallization inhibitors, characterized in that: The DNA nanostructure is formed by self-assembly of DNA sequences, and the self-assembly method comprises the following steps: providing a DNA sequence, wherein the DNA sequence is AAAAAAAAAA, AATTTTTTT, AAAATTTTTT, AAAAAATTTT, AAAAAAAATT, CCCCCCCCCC, or GGGGGGGGGG; The DNA sequence is prepared into an aqueous solution, and then an aqueous solution containing ferrous ions is added thereto. The strong coordination between the ferrous ions and the nitrogen and oxygen atoms on the DNA nucleobases drives the DNA to self-assemble. After the addition of the ferrous ions, the solution needs to be annealed at the melting point of the DNA for 2-3 hours. Finally, the product is centrifuged, washed, freeze-dried, and resuspended in the aqueous phase.

2. Use of DNA nanostructures as ice crystal recrystallization inhibitors, characterized in that: The DNA nanostructure is a spherical nucleic acid formed by a DNA single strand with a thiol group modified at the end and gold nanoparticles through a metal-thiol reaction; the particle size of the gold nanoparticles is 15-85nm, and the DNA single strand with a thiol group modified at the end is SH-AAAAAAAAAA.

3. Use of DNA nanostructures as ice recrystallization inhibitors, characterized in that: The DNA nanostructure is a spherical nucleic acid formed by a DNA single strand modified with a thiol group at the end and gold nanoparticles through a metal-thiol reaction; the particle size of the gold nanoparticles is 85 nm, and the DNA single strand modified with a thiol group at the end is SH-GGGGG, SH-GGGGGGGGGG or SH-GGGGGGGGGGGGGGG.

4. The method according to any one of claims 1 to 3, wherein: The DNA nanostructure is directly added to achieve ice crystal recrystallization inhibition.

5. The use according to claim 1, characterized in that The DNA nanostructure is used as an ice crystal recrystallization inhibitor and is added at a concentration of 0.5-5 mg / mL.

6. The use according to claim 1, wherein The DNA nanostructure is used as an ice crystal recrystallization inhibitor in cell freezing. Every 10 mg of ice crystal recrystallization inhibitor is added to 4×10 5 in a cell.

7. The use according to claim 2, characterized in that The particle size of the gold nanoparticles is 30-85 nm.

8. The use according to claim 2 or 3, characterized in that The DNA nanostructure is used as an ice crystal recrystallization inhibitor, and the gold nanoparticles are used as a quantitative standard, with an added concentration of 0.1-10 nM.

9. The use according to claim 2 or 3, characterized in that The DNA nanostructure is used as an ice crystal recrystallization inhibitor in cell cryopreservation. The core concentration of the spherical nucleic acid is used as a quantitative standard. When 0.4 nM ice crystal recrystallization inhibitor is added to 4×10 5 in a cell.

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