Cryoprotectant compositions containing DNA nanostructures and methods of use thereof

Abstract

The present invention relates to a composition for cryoprotection, the composition comprising: a nucleic acid structure comprising a scaffold nucleic acid that folds at a predetermined position and forms a plurality of chains, and a plurality of staple nucleic acids that form a double strand by binding to at least one of the chains and at least a portion of which has a complementary sequence to the scaffold nucleic acid; a linker bound to at least a portion of the nucleic acid structure; and a peptide bound to at least a portion of the linker, wherein the composition can increase cell viability during cryopreservation of cells and tissues and allows the texture of the food to be maintained when used for frozen food.

Description

Technical Field

[0001] The present invention relates to a composition for cryopreservation comprising a DNA nanostructure and a method for using the composition. Background Art

[0002] Antifreeze materials refer to compounds that, when present in a solution, can reduce or inhibit the formation of ice crystals in a solution exposed to a temperature below zero (0)°C. Currently, antifreeze materials include small molecules, synthetic polymers, and antifreeze proteins. In the case of currently used small molecules, very high effective concentrations (e.g., 60% or more) are required, thus sometimes causing tissue toxicity. In addition, in the case of naturally occurring proteins, it is difficult to purify and mass produce them, thus resulting in very high product price limitations.

[0003] Living organisms that live in extremely low temperature ("cryogenic") environments contain antifreeze proteins, which prevent body fluids from freezing, thereby protecting cells and tissues. Antifreeze proteins each have an inherent structure. However, a regular arrangement in a peptide called threonine is known to bind to specific surfaces of ice crystals and have the effect of inhibiting crystal growth.

[0004] DNA origami is a bottom-up fabrication technique for forming nanostructures using the complementary self-binding properties between four (4) unique bases of DNA, where the nanostructures are formed using very long single DNA strands called scaffold DNA, typically having 7000 bases or more, and multiple short staple DNAs that are used to conjugate the single-stranded DNA into specific shapes.

[0005] This DNA origami technology allows the production of two-dimensional / three-dimensional nanostructures with complex shapes with high precision within a few nanometers (nm), which cannot be prepared by conventional top-down manufacturing techniques. Through this technology, the application of nanostructures such as the precise arrangement of different nanomaterials at the desired position becomes possible. In addition, due to the use of biomolecules, the manufactured nanostructures can have very excellent biocompatibility.

[0006] Cryoprotectants act in a complex manner inside and outside cells. Specifically, structures absorbed inside cells inhibit the formation and growth of ice crystals in cells. On the other hand, structures present outside cells inhibit the formation and growth of ice cells around cells and prevent cells from being damaged by ice crystals outside cells and piercing the cell membrane.

[0007] Meanwhile, in the case of dimethyl sulfoxide (DMSO) as one of the cell cryoprotectants generally available in the art, it should be used in a large amount so that the mixing ratio of DMSO to the cell culture medium reaches about 10% (v / v). In addition, the above compound is a substance with cytotoxicity and has an adverse effect on cell viability (survival rate), so the problem is that it should be removed as soon as possible after thawing.

[0008] In addition, previous studies on DNA origami materials that demonstrated no cytotoxicity have been published, however, it is still not completely clear whether cytotoxicity is expressed or whether various types of cells are cytotoxic when the peptide having antifreeze ability is combined with the above DNA origami materials. Therefore, the above work needs to be evaluated for commercialization. Summary of the invention

[0009] [Problems to be Solved by the Invention]

[0010] An object of the present invention is to provide a composition for cryopreservation of cells or tissues, which comprises a DNA nanostructure.

[0011] In addition, another object of the present invention is to provide a composition for food cryopreservation, which comprises a DNA nanostructure.

[0012] In addition, another object of the present invention is to provide a method for cryopreservation of cells or tissues.

[0013] Furthermore, another object of the present invention is to provide a method for freezing and preserving food.

[0014] [Methods used to solve the problem]

[0015] In order to achieve the above-mentioned purpose, the following technical solutions are adopted in the present invention.

[0016] 1. A composition for cryopreservation, comprising:

[0017] A nucleic acid structure comprising: a scaffold nucleic acid folded at a predetermined position to form a multi-stranded structure; a plurality of first staple nucleic acids, wherein at least a portion of their sequence comprises a complementary sequence to the sequence of the scaffold nucleic acid, which binds to at least one strand of the scaffold nucleic acid to form a double strand; and a plurality of second staple nucleic acids, wherein at least a portion of their sequence is complementary to the sequence of the scaffold nucleic acid, but at least one end thereof is not complementary to the sequence of the scaffold nucleic acid, which binds to at least one strand of the scaffold nucleic acid to form a double strand and a single strand at its end;

[0018] a linker coupled to at least one single strand of the nucleic acid structure; and

[0019] An antifreeze peptide coupled to at least one of said linkers.

[0020] 2. The composition according to item 1 above, wherein the linker is DNA, RNA or PNA.

[0021] 3. The composition according to 1 above, wherein the linker has a length of 5 to 20 nucleotides.

[0022] 4. The composition according to 1 above, wherein the peptide has a length consisting of 1 to 10 amino acids.

[0023] 5. The composition according to 4 above, wherein the amino acid is alanine or threonine.

[0024] 6. The composition according to item 1 above, wherein the nucleic acid structure comprises two or more helical bundles and a surface suitable for contacting ice crystals.

[0025] 7. The composition according to 1 above, wherein the nucleic acid structure comprises a twisted or bent helical bundle.

[0026] 8. A composition for cryopreservation of cells or tissues, comprising the composition according to any one of 1 to 7 above.

[0027] 9. A composition for frozen preservation of food, comprising the composition according to any one of 1 to 7 above.

[0028] 10. A method for cryopreservation of cells or tissues, comprising exposing the test cells or tissues to a temperature below 0°C in the presence of the composition according to 8 above.

[0029] 11. A method for frozen preservation of food, comprising exposing a test food to a temperature below 0°C in the presence of the composition according to 9 above.

[0030] [Beneficial Effects]

[0031] The composition of the present invention has excellent cell and tissue cryopreservation ("cryopreservation") effects.

[0032] The use of the composition of the present invention can increase the cell viability of cells during cryopreservation.

[0033] The composition of the invention has excellent food freezing and preservation effect.

[0034] In freezing of food using the composition of the present invention, the food can maintain its original texture. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a diagram showing a state where a scaffold nucleic acid and a staple nucleic acid are bound to each other to form a structure.

[0036] Figure 2 is a diagram showing several nucleic acid origami structures, where A is a 4-helix bundle (rigid structure), B is a 4-helix bundle (flexible structure), C is a 24-helix patch (Rothemund square) structure, D is a 12-helix bundle (rigid structure), E is a 12-helix bundle (90° bent structure), and F is a 12-helix bundle (45° bent structure).

[0037] Figure 3 is a diagram showing the rigid structures of 4, 6 and 12 helix bundles (HB).

[0038] Figure 4 is a diagram showing a 4-helix bundle flexible structure and a 6-helix bundle flexible structure.

[0039] Figure 5 are diagrams showing origami structures with and without gaps.

[0040] Figure 6 is a schematic diagram showing a method for measuring the recrystallization inhibition (RI) effect.

[0041] Figure 7 and 8 are photographs and graphs showing the results of RI measurements of a buffer solution containing only MgCl 2 in pure distilled water.

[0042] Fig. 9 and 10 are photographs and graphs showing the results of RI measurement of a solution in which unstructured single-stranded DNA exists.

[0043] Fig.11 and 12 are photographs and graphs showing the results of RI measurements of scaffold DNA samples.

[0044] Fig.13 is a diagram showing the rigid structure and the flexible structure of the 4-helix bundle.

[0045] Fig.14 is a diagram showing the rigid structure and the flexible structure of the 6-helix bundle.

[0046] Fig.15 is a diagram showing a 24-helix patch structure.

[0047] Fig.16 is a diagram showing the solid structure and the bent structure of the 12-helix bundle.

[0048] Figures 17 to 20 are photographs and graphs showing the RI effect of a DNA origami structure.

[0049] Fig.21 The growth process of ice crystals with a rigid structure of a 6-helix bundle over time is shown.

[0050] Fig. 22 is a diagram showing the design of the DNA-PNA-peptide patch.

[0051] Fig.23 Shown are the results of agarose gel electrophoresis of the completed DNA-PNA-peptide patch.

[0052] Fig.24 The results of observing the actual appearance of the DNA origami structure used in the experiment by atomic force microscopy (AFM) are shown.

[0053] Fig.25 The results of agarose gel electrophoresis experiments are shown, in which the stability of the constructs (constructs without peptide patches) was tested in phosphate buffered saline (PBS) and cell culture medium (Dulbecco's Modified Eagle's Medium, DMEM), respectively.

[0054] Fig.26 Shown are the results of agarose gel electrophoresis experiments after stability testing of the structure (structure without peptide patch).

[0055] Fig. 27 The distribution of structures taken up inside HSC-3 cells or taken up to the outside of the cells is shown (purple: peptide patch, blue: cell nucleus).

[0056] Fig.28 Shown are the results of observing the protective ability of cells when frozen and then thawed, including DNA patches or controls (PBS buffer).

[0057] Fig.29 is a graph showing the cell viability of each origami structure as a function of incubation time (cryopreservation time was 24 hours, the same in all cases) compared to DMSO 10%.

[0058] Fig.30 is a graph showing the results of measuring cell viability after 24 hours of incubation and 1 month of cryopreservation.

[0059] Fig.31 The results of in situ confirmation of the cryopreservation effect of DNA origami are shown. DETAILED DESCRIPTION

[0060] Hereinafter, the present invention will be described in detail.

[0061] The present invention relates to a composition for cryopreservation ("cryopreservation composition") comprising a DNA nanostructure.

[0062] As used herein, cryopreservation refers to protecting cells, tissues and / or foods from freezing damage that may occur during the cryopreservation of cells, tissues and / or foods.

[0063] As used herein, freezing damage refers to damage that may occur in cells, tissues and / or foods when cells, tissues and / or foods are frozen. For example, damage to cell membranes may occur due to ice crystallization of water. In addition, when non-invasive cryoprotectants are used, they only affect the extracellular salt concentration, so the resulting high solute concentration may cause osmotic stress to the cell membrane. In addition, the high concentration of cryopreservatives required for vitrification may cause damage due to low water chemical potential or chemical toxicity. However, it is not limited to this.

[0064] In the present invention, cryopreservation can occur through the combined action of DNA nanostructures inside and outside cells and tissues. For example, the structure absorbed into the interior of the cell can inhibit the formation and growth of ice crystals inside the cell, while the structure present outside the cell can inhibit the formation and growth of ice crystals around the cell and prevent the cell from being damaged by ice crystals outside the cell and piercing the cell membrane. However, it is not limited to this.

[0065] The composition for cryopreservation of the present invention may include materials for protecting living tissues, cells or organisms and foods from freezing damage.

[0066] According to one embodiment of the present invention, the composition for cryopreservation can inhibit the formation or growth of ice crystals.

[0067] Ice crystals can grow by ice recrystallization, which refers to the process of growing from small ice crystals to larger ice crystals, and their growth occurs according to the Ostwald ripening mechanism. Ostwald ripening can occur in a dissolution-diffusion-refreezing or sublimation-diffusion-condensation mechanism. In other words, ice recrystallization does not occur due to ice crystals adhering to each other, but rather due to small ice crystals melting between crystals and diffusing toward large ice crystals, thereby becoming part of the large ice crystal when refreezing.

[0068] Inhibiting the growth of ice crystals refers to the act of preventing ice from forming, reducing the rate at which ice forms, preventing ice from recrystallizing, reducing the rate at which ice recrystallizes, or keeping the size of ice crystals small.

[0069] The composition for cryopreservation of the present invention can prevent freezing of cells or tissues. In addition, when cells are thawed after cryopreservation, cell viability can be improved.

[0070] The effect of preventing and inhibiting freezing of cells or tissues may vary depending on the degree of icephilic properties associated with the types and configurations of bases used to construct the DNA nanostructures contained in the composition.

[0071] The composition for cryopreservation of the present invention may include a nucleic acid structure.

[0072] The nucleic acid structure can be a nucleic acid origami structure.

[0073] The nucleic acid may be DNA, RNA or PNA, but is not limited thereto.

[0074] like Figure 1 As shown, the nucleic acid origami structure is formed by folding the scaffold nucleic acid ( Figure 1 The structure is formed by simultaneously binding multiple staple nucleic acids to a predetermined part of the DNA scaffold.

[0075] The scaffold nucleic acid is a single-stranded nucleic acid whose length can be appropriately selected depending on the length, size, shape, etc. of the structure to be formed, and an acid type with a length of about 7000 to 8000 nucleotides (nt) can be used. In a specific embodiment of the present invention, M13mp18 DNA with a length of 7,249 nt is used, but is not limited thereto. When the M13mp18 scaffold DNA is used to prepare a DNA structure, the molecular weight of the resulting structure is about 5 megadaltons, which is equivalent to about 10 -20 kg, but not limited to.

[0076] In the staple nucleic acid, at least a portion of its sequence has a complementary sequence to the scaffold nucleic acid, thereby allowing the scaffold nucleic acid to be folded or fixed at a specific position.

[0077] In the present invention, the staple nucleic acid may be a plurality of staple nucleic acids (hereinafter referred to as "first staple nucleic acid"), which bind to at least one strand of the scaffold nucleic acid to form a double strand. In addition, the staple nucleic acid may be a plurality of staple nucleic acids (hereinafter referred to as "second staple nucleic acid"), wherein at least a portion of its sequence is complementary to the sequence of the scaffold nucleic acid, but one end thereof is not complementary to the sequence of the scaffold nucleic acid, and it binds to at least one strand of the scaffold nucleic acid to form a double strand and a single strand at its end.

[0078] The first staple nucleic acid may have a complementary sequence to the scaffold nucleic acid sequence, and thus may bind to the scaffold nucleic acid to form a double strand.

[0079] The second staple nucleic acid may include a portion having a complementary sequence to the scaffold nucleic acid sequence and another portion that is not complementary to the scaffold nucleic acid at at least one end ("one end") on one side. For example, the one end may not be complementary to the scaffold nucleic acid, or both ends may not be complementary to the scaffold nucleic acid, however, it is not limited thereto.

[0080] In the present invention, the staple nucleic acid may include a plurality of separate first staple nucleic acids, a plurality of separate second staple nucleic acids, or a plurality of first and second staple nucleic acids together, but is not limited thereto.

[0081] The length of the staple nucleic acid may be appropriately selected based on the length, size, or shape of the structure to be formed, and for example, the length may range from 20 to 50 nt, but is not limited thereto.

[0082] In the present invention, at least one terminal portion of the second staple nucleic acid that is not complementary to the scaffold nucleic acid can be a portion designed so that by extending the length of a portion of the base sequence of the staple nucleic acid when designing the base sequence, at least one terminal of the base sequence of the staple nucleic acid is not complementary to the base sequence of the scaffold nucleic acid. When the second staple nucleic acid is bound to the scaffold nucleic acid, the above-mentioned at least one terminal can be exposed to one side of the nucleic acid origami, which is hereinafter referred to as the "handle". The nucleic acid origami structure is a specific structure formed by binding the staple nucleic acid to a specific position of the scaffold nucleic acid and folding the staple nucleic acid at the specific position, and the staple nucleic acid is designed so that the scaffold nucleic acid has this specific structure.

[0083] The design of the staple nucleic acid can be performed according to a conventional method, and for example, a design scheme such as caDNAno can be used, but is not limited thereto.

[0084] Nucleic acid origami structures can be prepared using conventional thermal annealing techniques.

[0085] This technology starts by heating the scaffold nucleic acid and staple nucleic acid as raw materials to a high temperature (e.g., 80°C to 95°C) so that all nucleic acid chains are in a single-stranded state. The nucleic acid chain has a melting temperature according to its base sequence, and above the melting temperature, the nucleic acid exists mainly as a single strand, and below the melting temperature, the nucleic acid exists mainly as a double strand. When the temperature of the reaction solution is slowly lowered, the nucleic acid chain begins to exist as a double strand while complementary binding, and in the nucleic acid origami, about 200 staple nucleic acids are bound to the designed position while synergistically binding, thereby producing a structure with a desired shape.

[0086] Since the respective staple nucleic acids have different base sequences, the binding times are slightly different from each other. However, because conventional thermal annealing techniques allow the temperature to be gradually reduced over a sufficient time (more than a few hours), conditions are formed in which all staple nucleic acids are sufficient to bind, and therefore a structure with a desired shape can be prepared regardless of the base sequence.

[0087] The scaffold nucleic acid folds at a predetermined position to form multiple chains, and the staple nucleic acid has a sequence designed to allow the scaffold nucleic acid to fold at a predetermined position. Therefore, the scaffold nucleic acid forms a predetermined structure, and the staple nucleic acid is combined with the scaffold nucleic acid by the above method.

[0088] The scaffold nucleic acid chain and the staple nucleic acid can be combined with each other to form a double strand (double helix), wherein the nucleic acid origami structure can have, for example, 2 to 50, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 12 or 4 to 12 helical bundles, and specifically can have 4 to 12 helical bundles, but is not limited thereto. Figure 2 and 4 Nucleic acid origami structures with different numbers of helix bundles are shown.

[0089] The linker can be combined with at least one single strand in the nucleic acid structure. Specifically, the linker can be combined with at least one of the single strands present at the end of the second staple nucleic acid, but is not limited thereto.

[0090] The linker may be any nucleic acid, at least a portion of which has a complementary base sequence at the handle, and may include a nucleic acid chain such as DNA, RNA, PNA, etc., but is not limited thereto. The linker may be a chain to which a functional material such as phosphorus, peptide, etc. is additionally attached, but is not limited thereto.

[0091] The connector is combined with at least one of the above handles, and can be combined with one or more or all of the handles, but is not limited thereto.

[0092] The length of the linker can be appropriately selected based on the length of the handle, the configuration of the bases, and the length, size, shape, etc. of the structure to be formed. For example, the length of the linker can range from 5 nucleotides (nt) to 50 nt, specifically, 5 nt to 30 nt, but is not limited thereto.

[0093] In the present invention, the antifreeze peptide may be a peptide bound to at least one of the above-mentioned linkers.

[0094] The antifreeze peptide may be bound to at least a portion of the linker. At least a portion of the linker refers to one or more chains in a single chain of a nucleic acid structure, or all or part of a complete base sequence of one of the linkers. For example, the peptide may be bound to one end of the linker or both ends of the linker. The binding between the linker and the antifreeze peptide may be carried out by hydrogen bonds, covalent bonds, peptide bonds, etc., but is not limited thereto.

[0095] In one embodiment of the present invention, a linker (e.g., a peptide nucleic acid chain (PNA)) having a complementary base to the base sequence of the handle portion can be designed, and a peptide can be bound to at least one of the linkers (hereinafter referred to as a "probe"). When a nucleic acid origami structure is formed in an aqueous solution, the probe can be bound to the handle as part of the design (see Fig.24 ).

[0096] As used herein, the peptide refers to a peptide having an antifreeze effect. The effect of inhibiting ice crystal growth may vary depending on the degree of ice-philic properties based on the type of peptide and the density of peptide arrangement.

[0097] In one embodiment of the present invention, the peptide may include a single or combined type of amino acid polymer. The amino acid may have an antifreeze effect. For example, the amino acid may include alanine or threonine, but is not limited thereto.

[0098] The length of the amino acid polymer can be appropriately selected based on the length and base configuration of the linker, the length or base configuration of the exposed single strand, the length and size, shape of the structure to be formed, etc. For example, the length of the amino acid polymer can range from 1 to 20 amino acids, 1 to 10 amino acids, or 2 to 10 amino acids, but is not limited thereto.

[0099] In the nucleic acid origami structure of the present invention, the antifreeze peptides may be arranged at predetermined intervals. In one embodiment of the present invention, an arrangement of antifreeze peptides may be formed on one side of the nucleic acid origami structure.

[0100] Since the origami structure has more handles, the number of antifreeze peptides attached to the handles can also be increased, thereby improving the antifreeze effect. In one embodiment of the present invention, an origami structure having 96 antifreeze peptides arranged at intervals of 16 nt is present in the nucleic acid origami sheet structure (in Fig. 22 In the lateral direction of one side of the origami structure in which the antifreeze peptides are arranged at all the handle parts), the origami structure with 48 antifreeze peptides arranged at intervals of 32 nt in the lateral direction showed higher cell viability (see Fig.29 ).

[0101] The structure of the present invention can be absorbed into the interior of the cell or adhered to the outer wall of the cell to encapsulate the cell. The material absorbed into the interior of the cell can inhibit the formation and growth of ice crystals generated inside the cell during the freezing / thawing process of the cell. In addition, the material absorbed into the interior of the cell can prevent and inhibit cells and tissues from being frozen. In addition, when the cell is thawed after cryopreservation, the cell viability can be improved.

[0102] The structure adhered to the outside of the cell can inhibit the formation and growth of ice crystals generated around the cell, inhibit ice crystals from penetrating into the cell, and protect the cell membrane from damage. In addition, the structure adhered to the outside of the cell can prevent and inhibit cells and tissues from being frozen and prevent damage to them. In addition, when cells are thawed after cryopreservation, cell viability can be improved.

[0103] The structure used in the present invention may have a weight / volume ratio (percentage) of 0.001% to 0.03% in aqueous solution and 0.001% to 0.003% in cell culture medium.

[0104] Compared with the permeable small molecules used for existing tissue preservation that require a high weight / volume ratio of up to several tens of percent, the nucleic acid structure of the present invention can have an ice crystal growth inhibition effect even at a relatively low weight / volume ratio, and can further exhibit an antifreeze effect on cells or tissues. In addition, when cells are thawed after cryopreservation, cell viability can be improved.

[0105] In this regard, the nucleic acid origami structure according to the present invention may include a twisted or bent helical bundle.

[0106] For example, the direction in which the helical bundle is twisted can be controlled by inserting or deleting bases, and the bundle can be twisted to the right by inserting bases into the helix, or can be twisted to the left by deleting bases. The degree of twisting can be controlled by the number of bases. The number of bases to be inserted or deleted can be one or two per 21 base lengths of two turns of the helix, and even if the number of bases exceeds the above range, there is no particular limitation as long as there is no problem in maintaining the structure.

[0107] Furthermore, for example, a base is inserted into one of the plurality of helices without inserting the base into the other helices, or the base is deleted so that the lengths between the helices differ, so that the helical bundle may be curved.

[0108] In addition, in terms of inhibiting ice crystal formation or growth, i.e., ice recrystallization, the nucleic acid origami structure may include a surface that can contact the ice recrystallization surface. The ice recrystallization inhibition effect can also be maximized by making the surface of the nucleic acid origami structure contact the ice recrystallization surface in a large area in the form of a plane rather than a line. In addition, the nucleic acid origami structure can be a large area in a planar form, and the antifreeze effect of cells or tissues can be maximized.

[0109] In addition, the present invention relates to a composition for cryopreservation of cells or tissues, comprising the above composition.

[0110] When cells or tissues are cryopreserved, during the process of thawing the cryopreserved cells or tissues for subsequent use, cell membranes may be damaged by ice recrystallization, thereby causing cell and tissue damage due to cellular dehydration. Organisms living in low-temperature environments may be more susceptible to damage from ice recrystallization.

[0111] The nanostructure included in the composition of the present invention can be absorbed into the interior of cells and tissues, or adhere to the outer cell wall of cells and tissues, so as to encapsulate food. The material absorbed into the interior of cells and tissues can suppress the formation and growth of ice crystals produced in the process of freezing / thawing cells and tissues. In addition, the material absorbed into the interior of cells and tissues can prevent and suppress food from being frozen. In addition, the structure that adheres to the exterior of cells and tissues can prevent and suppress cells and tissues from being frozen, and further prevents damage to it.

[0112] The composition of the present invention is applicable to all cells that are usually frozen for preservation, and may include, for example, prokaryotic cells; eukaryotic cells; microorganisms; animal cells; cancer cells, sperm; eggs; stem cells, including adult stem cells, embryonic stem cells and dedifferentiated stem cells; blood cells, including umbilical cord blood, white blood cells, red blood cells and platelets; and tissue cells, including kidney cells, liver cells and muscle cells, but are not limited thereto.

[0113] Furthermore, the composition of the present invention is applicable to all tissues that are usually frozen for preservation, and may include, for example, all tissues such as cornea, kidney, heart, small intestine, pancreas, lung, liver, etc., but is not limited thereto.

[0114] The composition of the present invention may further include a preservative solution for cryopreservation of cells and / or tissues. The preservative solution may include, for example, water, saline, phosphate buffered saline (PBS), various cell culture media, but is not limited thereto.

[0115] The composition of the present invention may include 0.001% to 0.5% (w / v) of the nucleic acid structure, and specifically 0.01% to 0.03% (w / v), but is not limited thereto.

[0116] The permeable small molecules used in current tissue preservation methods require a high weight / volume ratio of up to several tens of percent, while the nucleic acid origami structure of the present invention can have an ice recrystallization inhibition effect even at extremely low contents, which may be more advantageous for the preservation of cells and tissues after freezing. In addition, when cells are thawed after cryopreservation, cell viability can be improved.

[0117] Furthermore, the present invention relates to a method for cryopreservation of cells or tissues, the method comprising exposing the subject cells or tissues to a sub-zero temperature in the presence of the composition.

[0118] When subject cells or tissues are frozen in the presence of the composition, damage to the cells or tissues can be prevented by inhibiting ice recrystallization upon subsequent thawing.

[0119] Furthermore, the present invention relates to a composition for frozen preservation of food comprising the above composition, and a method for frozen preservation of food, the method comprising exposing a test food to a temperature below zero (0° C.) in the presence of the above composition.

[0120] The composition of the present invention can be applied to all frozen foods, and when used, the degradation of the texture of the food can be minimized when it is subsequently thawed.

[0121] For example, the structure of the present invention can be absorbed into the food or adhered to the outer wall of the food to encapsulate the food. The material absorbed into the food can inhibit the formation and growth of ice crystals generated inside the food during the freezing / thawing process of the food. In addition, the material absorbed into the food can prevent and inhibit the food from being frozen. In addition, the structure adhered to the outside of the food can prevent and inhibit the food from being frozen, and further prevent damage to it.

[0122] Hereinafter, the present invention will be described in detail with reference to Examples.

[0123] Example

[0124] 1. Experimental identification of the inhibitory effects of cations and DNA structure on ice recrystallization

[0125] 1.1 Experimental method of ice recrystallization

[0126] The splash cooling experimental technique known in the art was used to measure the RI effect (see Figure 6). First, a cover glass is placed on a metal plate pre-cooled with liquid nitrogen (the temperature of the cover glass surface is about -150°C), and 20 μl of a solution containing a material for measuring the RI effect dispersed therein is dripped at a height of about 1.5 m. As soon as the solution contacts the cover glass, it spreads thinly and freezes while being rapidly cooled, and many very small ice crystals are formed due to the rapid cooling. The cover glass is then transferred to a cold stage with a drilled hole through which the ice crystals can be observed with an optical microscope. The cold stage is maintained at -6°C, and the state in which the ice crystals grow into small crystals that gradually fuse is observed for 30 minutes. Thereafter, the size of the ice crystals is measured to compare how much less ice crystals have grown compared to the control (a sample containing only the solution without the frozen control material).

[0127] 1.2. Observation of RI effect in aqueous solutions containing cations and unstructured DNA samples

[0128] First, RI measurement experiments were performed on a buffer solution containing only MgCl2 in pure distilled water, and it was confirmed that ice recrystallization was suppressed as the ion concentration increased (see Figure 7 and 8 ). Based on the results of this experiment, the MgCl2 concentration of the reference buffer solution was set to 5 mmol (mM) in subsequent experiments. The RI performance of the sample was measured by comparing the relative RI performance of the sample with the reference buffer, which means that the smaller the value, the higher the RI performance.

[0129] Next, the RI measurement experiment was performed on a solution containing DNA in an unstructured single-stranded state. The DNA concentration used in the experiment was set to 50 to 200 ng / uL, which is similar to the concentration of DNA structures prepared by conventional manufacturing processes. As a result of measuring the RI performance of a sample in which 169 types of stapled DNAs forming a 6-helix bundle rigid structure were mixed in a single-stranded state, it was observed that it had a value of 0.87 to 1.0, and there was almost no ice recrystallization inhibition effect compared to the buffer solution (see Fig. 9 and 10 ). In other words, the experiment confirmed that although the DNA chain has aggregated Mg 2+ ions, but it is impossible to maintain Mg 2+ ions, unless they form a structure with a constant shape and rigidity in aqueous solution, so that almost no RI effect occurs at the macroscopic level.

[0130] In addition, the RI value of the scaffold DNA sample performed in the same manner was measured to be as high as about 0.7 (see Fig.11 and 12). The reason why the scaffold DNA has a slight RI performance compared with the staple DNA is considered to be due to the characteristics of the long scaffold DNA, which forms a secondary structure in which the entire region does not exist in a single-stranded state, but some parts are combined into double strands in a complementary manner to each other.

[0131] 1.3. Design of structured DNA origami

[0132] After that, experiments were conducted on DNA structures in which DNA chains were tightly connected to each other, thereby having a constant cross-sectional shape and rigidity. Nine types of structures designed as examples of DNA origami technology include: (1) 4-helix bundle rigid structure; (2) 4-helix bundle flexible structure (gap 5nt); (3) 6-helix bundle rigid structure; (4) 6-helix bundle flexible structure (gap 3nt); (5) 6-helix bundle flexible structure (gap 5nt); (6) 24-helix patch; (7) 12-helix bundle rigid structure; (8) 12-helix bundle bent structure (90°); and (9) 12-helix bundle bent structure (45°).

[0133] The design approach for each structure is as follows.

[0134] (1) Rigid structure of 4-helix bundle and (2) Flexible structure of 4-helix bundle (gap 5 nt)

[0135] Structures (1) and (2) have a cross section consisting of four double-stranded DNA strands, one side of which is packed into a square of approximately 4.5 nm ( Fig.13 ). They have a structure based on the repetition of the same form of connection over a length of about 192 nt, and each has a total length of about 600 nm. The rigid structure of (1) is designed so that the opposite ends (5' end and 3' end) of all stapled DNAs present in the structure are in direct contact with each other, while the flexible structure of (2) is composed of ssDNA, in which the gap between the opposite ends of 150 adjacent stapled DNAs is 5 nt.

[0136] (3) 6-helix bundle rigid structure, (4) 6-helix bundle flexible structure (gap 3 nt) and (5) 6-helix bundle flexible structure (gap 5 nt)

[0137] Structures (3), (4), and (5) have cross-sections consisting of six double-stranded DNA strands packed into a hexagonal shape ( Fig.14 ). They have a structure based on a repeating connection of the same form over a length of about 168 nt, and each has a total length of about 400 nm. The rigid structure of (3) is designed so that the opposite ends of all stapled DNAs present in the structure are in direct contact with each other, while the flexible structures of (4) and (5) are composed of ssDNA, in which the gaps between the opposite ends of 169 adjacent stapled DNAs are 3 nt and 5 nt, respectively.

[0138] (6) 24 spiral patches

[0139] Structure (6) has a cross section consisting of 24 double-stranded DNAs arranged in a straight line to form a sheet (see Fig.15 ). The structure has a length of about 90 nm (256 nt) and a width of about 55 nm. This structure is designed so that all ends of the 192 stapled DNAs present in the structure are in direct contact with each other.

[0140] (7) 12-helix bundle rigid structure, (8) 12-helix bundle bent structure (90°) and (9) 12-helix bundle bent structure (45°)

[0141] Structures (7), (8), and (9) have a cross-section consisting of 12 double-stranded DNA strands packed into a hexagon (see Fig.16 ). The rigid structure of (7) was designed so that all ends of the 180 stapled DNAs were in direct contact with each other. Structures (8) and (9) had a flexible region of 42 nt in length by removing 11 stapled DNAs present in the middle of the structure, and formed a shape in which the lengths of the DNAs connecting the opposite ends of the structure were shortened to 357 nt and 189 nt, respectively, and the central portions thereof were bent by 90° and 45°, respectively.

[0142] 1.4 Preparation of DNA origami and observation of RI effect

[0143] Thereafter, the ice recrystallization inhibition effect of a DNA structure constructed to have a constant cross-sectional shape and size was confirmed. The method for preparing the origami structure is as follows: after mixing the raw materials and the buffer, the mixture is then heated to 80°C, the temperature is lowered to 65°C within 1 hour, and then a thermal annealing technique is performed to lower the temperature to 25°C at 1°C per hour. In order to separate the structure prepared by the thermal annealing technique, a centrifugal purification filter manufactured by Amicon was used to filter the unreacted material. The concentration of the structure after purification was measured using absorbance at a wavelength of 260nm, and then the structure was diluted with a buffer to reach a target concentration.

[0144] The shape of the structure prepared according to the above design was measured by AFM (see Fig.17 , 18 As a result of the RI experiment on at least nine types of DNA structures having at least two types of different densities, a maximum RI value of 0.53 was measured (see Fig. 20 ). The remaining samples showed higher RI performance than the 4-helix bundle with the narrowest cross-sectional area (samples 1 and 2), and the higher the concentration of the structure, the higher the RI performance overall. This is believed to be because Mg 2+Ions can be sufficiently concentrated near the structure so that RI performance can be exhibited when the cross-sectional area is at a certain level, and as the number of structures in the aqueous solution increases, the area capable of suppressing ice recrystallization also increases. Regarding the 6-helix bundle rigid structure, as a result of observing the ice growth process after 30 minutes, it was observed that after about 2 hours, ice grew to a size similar to that of ice crystals in a buffer solution without the material (see Fig.21 ).

[0145] Through a series of experiments, it was demonstrated that a composition including a DNA structure in which cations are enriched can exert a higher ice recrystallization inhibition effect than DNA that does not form the structure.

[0146] 2. Fabrication of DNA-PNA-peptide origami structures

[0147] Design of DNA-PNDA-peptide origami structures

[0148] DNA-PNA-peptide structures with scaffold DNA, staple DNA, handle, Cy3 dye, and PNA-peptide binding sites were designed (see Fig. 22 ).

[0149] 2.2. Fabrication of DNA-PNA-peptide origami structures (see Fig.23 )

[0150] To make a structure with a PNA-peptide probe attached, all nucleic acid materials used to build the structure, i.e., the scaffold DNA, the staple DNA including the handle, and the PNA-peptide, are mixed simultaneously. Thereafter, a DNA-PNA-peptide structure is formed in which the DNA-peptide probe is attached to the handle site of the DNA origami, according to the same thermal annealing process as in the fabrication of the DNA origami structure. The formed DNA-PNA-peptide structure is subjected to the same purification process as for the DNA origami structure.

[0151] 3. Confirmation of DNA structure's ability to protect cells

[0152] 3.1. Experimental materials and conditions

[0153] This experiment was performed using HSC-3 cells as cancer cells. The concentrations of the nucleic acid origami structures used in the experiment were 100 ng / uL and 200 ng / uL, respectively. When the peptide probe was attached, the structure was used at a concentration of 300 ng / uL. The final material for cell experiments was prepared at a concentration of 10 ng / uL to 30 ng / uL by mixing the cell culture medium and the cryopreservation material in a volume ratio of 9:1.

[0154] First, the nucleic acid origami structure was subjected to the measurement of the cryoprotective effect on the structure only in the absence of peptide probes. Thereafter, PNA probes attached with antifreeze peptides such as alanine and threonine were used to carry out experiments to measure the cryoprotective ability of DNA-peptide combination materials.

[0155] The nucleic acid origami used to determine the cell protection ability had the following shapes: (1) 4-helix bundle structure; (2) 6-helix bundle structure; (3) 12-helix bundle structure; and (4) 24-helix patch structure. In addition, the shape of the structure formed according to the above design process was confirmed by AFM (see Fig.24 ).

[0156] 3.2. Confirmation of structural stability in saline and cell culture medium

[0157] To confirm whether the DNA origami structure can maintain its structure during interaction with cells, the DNA structure was mixed with 10% phosphate buffered saline (PBS) and DMEM cell culture medium at a ratio of 9:1 and then incubated at 37°C for up to 8 hours.

[0158] The incubation was completed at 1, 2, 4, and 8 hours, and the appearance of the DNA structure was subsequently observed by AFM and agarose gel electrophoresis to confirm its structural stability (see Fig.25 and 26 ).

[0159] 3.3. Confirmation of Cellular Uptake

[0160] Whether the prepared patch structure was absorbed into / outside of the cells and attached to them was monitored by confocal fluorescence microscopy (see Fig. 27 ).

[0161] As fluorescent markers, Hoechst 33258 was used for cell nucleus staining, and Cy3 was used for DNA origami material. The cell nucleus was stained in blue, and the location of the origami was indicated in magenta.

[0162] The solution containing the DNA-PNA-peptide patch was mixed in a cell culture medium at a mixing ratio of 10%, followed by incubation. As a result of observation immediately after incubation, 1 hour later, and 2 hours later, it was confirmed that the amount of the patch present inside / outside the cells gradually increased with the incubation time.

[0163] 3.4. Confirmation of cell viability

[0164] 3.4.1. Confirmation of changes in cell viability by microscopy when thawing immediately after freezing

[0165] First, the cryoprotective ability of a typical DNA patch structure was confirmed by microscopy. Then, after the cells were mixed with 10% PBS and the DNA patch structure and incubated, the mixture was subjected to freeze-thaw as in the RI experiment, and then the survival of the cells was confirmed from the image. As a result of the experiment, it was confirmed that all cells in the PBS state were almost destroyed, but quite a few cells mixed with the DNA patch structure survived (see Fig.28 ).

[0166] 3.4.2. After 24 hours of cryopreservation, cell viability was confirmed by MTT assay upon thawing.

[0167] When cells were incubated at 37°C in a cell culture medium containing various DNA structures and various PNA-peptides bound thereto, cell viability over time was demonstrated (see Fig.29 ).

[0168] For the samples including the patch (PNA-peptide probe), the highest cell viability was found when incubated for 2 hours. Among them, when the DNA structure containing the probe having five (5) threonines (Thr×5) at a concentration of 300 mg / ul was incubated at liquid nitrogen temperature for 2 hours and then thawed after cryopreservation for 24 hours, it showed the highest cell viability among all the samples.

[0169] Meanwhile, for the bundle-type structure, cell viability tended to increase with incubation time.

[0170] Cell viability was compared and determined between origami samples and the cryoprotectant DMSO 10% (see Fig.29 ).

[0171] The patch structure in the form of a layer exhibits the most excellent cell protection ability because the above structure has the widest cross-sectional area relative to the material volume and flexibility, whereby the structure can effectively interact with the cell membrane and thus exhibit cryoprotective ability.

[0172] The material in which the alanine and threonine peptides were attached to the patch structure was confirmed to have much higher cryoprotective ability. From this result, it was confirmed that the peptides with cryoprotective ability showed excellent ice crystal formation and growth inhibition performance inside and around cells. In the case of the material using 96 probes attached to threonine, it was confirmed that this material had better performance than DMSO 10%, which is a commercially available cryoprotectant in the art.

[0173] 3.4.3. Confirmation of cell viability upon thawing after long-term cryopreservation

[0174] Based on the experimental conditions of 2 h incubation showing the highest cell viability, to confirm cell viability in long-term cryopreservation beyond 24 h, cells were cryopreserved at liquid nitrogen temperature for 1 month, then thawed and cell viability was subsequently measured (see Fig.30 ).

[0175] As a result, it was confirmed that the DNA structure with the Thr×5 probe had the highest cell viability, which was basically the same as the result obtained after 24 hours of cryopreservation. In addition, it was also confirmed that the structures at 200ng / ul and 300ng / ul, respectively, still had higher performance compared to the DMSO10% control. From this result, it can be understood that the cryoprotectant using the origami structure can be used for long-term cryopreservation.

[0176] 3.5. In situ RI and confirmation of cell status

[0177] Similar to the experimental method described in Example 1.1, an experiment for determining the RI performance of materials inside cells was performed at -6°C for 30 minutes. The nucleus part (blue staining), cytoplasm part (green), and cell membrane part (red) of the cells were stained separately, and their characteristics related to temperature changes were observed by fluorescence microscopy. For the control (PBS buffer), the nucleus of the cells after low-temperature freezing was damaged and had an irregular shape, so it was difficult to find the nucleus. For the cytoplasm, the size of the ice crystals inside the cells increased rapidly during the RI measurement, and very few ice crystals remained after thawing. Therefore, it can be confirmed that most of the cytoplasm disappeared due to cell damage. In addition, it can be confirmed that the cell membrane was initially distorted and then destroyed (see Fig.31 ).

[0178] In the case of the experimental group in which the patch structure at a concentration of 10% was mixed with the cell culture medium, it was confirmed that the nuclei of the cells subjected to cryogenic freezing remained similar to the initial stage. In addition, the size of the ice crystals inside the cells observed through the cytoplasm was much smaller than that of the control, and therefore, it was confirmed that the material had the effect of inhibiting the growth of ice crystals even inside the cells. In addition, even when thawing after freezing, the brightness of the cytoplasm was maintained, and therefore, it was confirmed that the damage to the cells was very small. In addition, because the fluorescent image of the cell membrane was similarly maintained even after freezing and thawing, the cell cryoprotective effect of the material was observed.

Claims

1. A composition for cryopreservation, comprising: Nucleic acid structures, including: scaffold nucleic acid that folds at predetermined positions to form multiple strands; a plurality of first staple nucleic acids, wherein at least a portion of their sequence comprises a complementary sequence to the scaffold nucleic acid sequence, and which bind to at least one strand of the scaffold nucleic acid to form a double strand; and a plurality of second staple nucleic acids, wherein at least a portion of their sequence is complementary to the sequence of the scaffold nucleic acid, but at least one end thereof is not complementary to the sequence of the scaffold nucleic acid, and which bind to at least one strand of the scaffold nucleic acid to form a double strand and a single strand at its end; a linker coupled to at least one single strand of the nucleic acid structure; and an antifreeze peptide coupled to at least one of said linkers, wherein the nucleic acid structure comprises 4 to 40 helical bundles, and The composition comprises 0.01% to 0.5% of the nucleic acid structure in terms of weight / volume percentage.

2. The composition of claim 1, wherein the linker is DNA, RNA or PNA.

3. The composition of claim 1, wherein the linker has a length of 5 to 20 nucleotides. The composition according to claim 1 , wherein the peptide has a length consisting of 1 to 10 amino acids. The composition according to claim 4 , wherein the amino acid is alanine or threonine.

6. The composition of claim 1, wherein the nucleic acid structure comprises a surface suitable for contact with ice crystals.

7. The composition of claim 1, wherein the nucleic acid structure comprises a twisted or bent helical bundle.

8. A composition for cryopreservation of cells or tissues, comprising the composition according to any one of claims 1 to 7.

9. A composition for frozen preservation of food, comprising the composition according to any one of claims 1 to 7.

10. A method for cryopreservation of cells or tissues, comprising exposing a subject cell or tissue to a temperature below 0°C in the presence of the composition according to claim 8.

11. A method for frozen preservation of food, comprising exposing a subject food to a temperature below 0°C in the presence of the composition according to claim 9.

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