RNA origami-based programmable extracellular matrix-mimetic materials for tissue engineering

Abstract

To provide an extracellular matrix-mimetic material dynamically formed and evolves in vivo, overcoming the limitations of conventional static biomaterials.SOLUTION: Provided is an extracellular matrix mimetic material being a programmable material in which RNA origami tiles self-assemble into a three-dimensional mesh network, which are transcribed from a DNA template by RNA polymerase and, simultaneously with transcription, fold up to self-assemble. The extracellular matrix mimetic material allows mechanical properties (persistence length 0.8 to 3.4 Mm) thereof to be precisely controlled by modifying the RNA sequence, and functional elements such as growth factor binding aptamers and cell adhesion motifs to be incorporated. Use of promoters that respond to external stimuli allows for achieving time-varying properties, and a degradation rate can be adjusted by RNA modification. The extracellular matrix mimetic material is provided as a hydrogel containing DNA template, RNA polymerase, nucleotide precursors, and magnesium ions and is applicable in tissue engineering fields such as wound healing, cartilage / bone / nerve regeneration, and vascularization promotion.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to programmable RNA nanostructures that mimic the extracellular matrix (ECM) for biomaterials engineering, particularly tissue engineering and regenerative medicine. More specifically, the present invention relates to extracellular matrix mimetic materials that are dynamically formed in vivo using RNA origami nanotubes and networks formed by the folding of RNA transcribed from a DNA template simultaneously with transcription. The present invention further relates to a biocompatible hydrogel composition comprising a DNA template, RNA polymerase, nucleotide precursors, and magnesium ions for generating such RNA origami-based extracellular matrix mimetic materials, as well as methods for manufacturing and using the same.

Background Art

[0002] In tissue engineering, the extracellular matrix (ECM) plays an important role in controlling cell behavior, differentiation, and tissue formation. Natural ECM is a complex three-dimensional network composed of structural proteins such as collagen, elastin, fibronectin, laminin, and non-structural proteins such as proteoglycans and glycosaminoglycans. These components not only provide structural support but also provide biochemical and mechanical signals that regulate cell functions such as cell adhesion, migration, proliferation, and differentiation.

[0003] Natural ECM is not a static structure but dynamically changes according to the developmental stage of the tissue and the pathological condition. For example, during the wound healing process, a temporary fibrin matrix is formed in the initial inflammatory phase, followed by the production of collagen by fibroblasts in the proliferative phase, and finally a mature collagen network is formed in the final remodeling phase. Such dynamic changes are essential for proper tissue regeneration.

[0004] Current synthetic ECM scaffolds are mainly composed of materials derived from natural sources such as collagen, fibrin, hyaluronic acid, or synthetic polymers such as polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL). Although these materials have achieved some success in tissue engineering and regenerative medicine, they have several important limitations.

[0005] Materials derived from natural sources are biocompatible and contain cell recognition sites, but they have problems such as batch - to - batch variation, limited mechanical properties, and lack of a controlled degradation profile. For example, collagen scaffolds may have significantly different mechanical properties and biochemical compositions between different batches, making it difficult to obtain reproducible results. Also, natural - derived materials often need to be chemically cross - linked to provide the mechanical properties required for a specific tissue type, which may reduce biocompatibility.

[0006] On the other hand, synthetic polymers provide reproducibility and controllable mechanical properties, but often lack biological recognition sites and require additional modifications to promote cell adhesion and bioactivity. For example, PEG - based hydrogels exhibit excellent mechanical properties and a controllable degradation profile, but lack cell adhesion sites and thus need to be modified with bioactive molecules such as RGD peptides. Also, the degradation products of synthetic polymers may, in some cases, cause local inflammatory reactions.

[0007] On the other hand, synthetic polymers provide reproducibility and controllable mechanical properties, but often lack biological recognition sites and require additional modifications to promote cell adhesion and bioactivity. For example, PEG - based hydrogels exhibit excellent mechanical properties and a controllable degradation profile, but lack cell adhesion sites and thus need to be modified with bioactive molecules such as RGD peptides. Also, the degradation products of synthetic polymers may, in some cases, cause local inflammatory reactions.

[0008] In the field of nucleic acid nanotechnology, the development of self-assembled nanostructures using DNA and RNA is progressing. DNA origami has been established as a technique for forming complex two-dimensional and three-dimensional structures using long scaffold strands and a large number of short staple strands. These structures can be designed with nanometer-scale precision, and various functional molecules can be placed at specific positions. DNA origami has been applied in fields such as biosensing, drug delivery, and molecular computing.

[0009] However, DNA origami has some limitations regarding in vivo applications. In particular, the formation of DNA origami usually requires a thermal annealing process (gradually cooling from 90°C), which is not achievable under physiological conditions. Also, since DNA origami is composed of multiple components (scaffold strands and staple strands), precise adjustment of the amount ratio of these components is necessary, and spontaneous formation in vivo is difficult. Furthermore, in many cases, chemical modifications (e.g., membrane anchoring with cholesterol) are required to confer functionality, and generating these structures from scratch in vivo becomes even more complex.

[0010] On the other hand, RNA origami is a relatively new technique in which single-stranded RNA folds during transcription to form complex three-dimensional structures. Compared with DNA origami, RNA origami has the following advantages: (1) Since it is formed from a single strand, precise adjustment of the amount ratio of multiple components is not required; (2) Since it folds simultaneously with transcription, additional processes such as thermal annealing are not required; (3) RNA is naturally biodegradable and suitable for in vivo use; (4) Functional RNA sequences such as aptamers can be easily incorporated.

[0011] Recent studies have shown that RNA origami tiles can fold during transcription and further self-assemble to form micrometer-scale nanotubes. These nanotubes can have their mechanical properties (rigidity or flexibility) adjusted by sequence design and can incorporate aptamers with specific functions. Furthermore, these RNA origami structures can be expressed within liposomes, indicating the potential for in vivo formation.

[0012] Of particular note is that RNA origami nanotubes exhibit structural properties similar to those of the natural cytoskeleton (microtubules). Microtubules are hollow cylindrical structures that play important functions such as maintaining cell morphology, intracellular transport, and cell division. RNA origami nanotubes have a similar hollow cylindrical structure to microtubules, and their rigidity (persistence length) can be adjusted by sequence design. Also, RNA origami nanotubes show the ability to form bundles under high magnesium concentrations, which is similar to the properties of natural cytoskeletal proteins.

[0013] However, attempts have not been made so far to apply these RNA origami technologies as extracellular matrix mimicking materials for tissue engineering. The properties of RNA origami (self-organization during transcription, adjustment of mechanical properties by sequence design, incorporation of functional aptamers) are ideal for the development of dynamic and responsive extracellular matrix mimicking materials, but this potential has not yet been explored.

Prior Art Documents

Non-Patent Documents

[0014]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0015] The object of the present invention is to solve the following problems: 1. Provision of an extracellular matrix-mimicking material that is dynamically formed and evolves over time: Development of a material that is dynamically formed in response to the in-vivo environment and whose properties change in response to cell growth and tissue development, unlike conventional static biomaterials. Specifically, there is a need for a material that can gradually release different growth factors according to the developmental stage of the tissue, exhibits different cell adhesion properties, and has different mechanical properties. 2. Development of an ECM-mimicking material capable of precisely controlling the presentation of mechanical properties and biochemical signals: Realization of a material whose mechanical properties (rigidity or flexibility) can be precisely controlled by sequence design and whose biochemical properties such as binding affinity with specific growth factors and cell adhesiveness can be programmed. In particular, there is a need for a material that has mechanical properties suitable for different tissue types (bone, cartilage, skin, nerve, etc.) and can present biochemical signals specific to each tissue. 3. Realization of an ECM-mimicking system composed of minimal components and capable of self-organizing in vivo: Development of a material that can be spontaneously formed in vivo without the need for complex manufacturing processes or non-physiological conditions such as heat annealing. In particular, there is a need for a system in which a complex three-dimensional network self-organizes from minimal components such as DNA templates, RNA polymerases, and nucleotide precursors. 4. Provision of a nanostructured ECM-mimicking material having biocompatibility and biodegradability: realization of a material that minimizes the immune response and degrades at an appropriate rate according to tissue growth. In particular, there is a need for a material that utilizes the natural biodegradability of RNA while enabling control of its degradation rate. 5. Development of an injectable form of ECM-mimicking material that can conform to a complex three-dimensional tissue structure: provision of a material that forms a three-dimensional network in situ after injection in a liquid state. In particular, there is a need for a material that can conform to a tissue defect site with a complex shape and provide structural support in situ. 6. Development of a smart ECM-mimicking material whose properties change in response to external stimuli: realization of a material whose mechanical and biochemical properties change in response to external stimuli such as light, temperature, pH, and specific small molecules. In particular, there is a need for a system that enables a physician to control the properties of the material during the treatment process.

Means for Solving the Problems

[0016] To solve the above problems, the present invention provides the following technical means:

[0017] A first aspect of the present invention is a programmable RNA origami-based extracellular matrix-mimicking material in which RNA oligomers self-assemble to form a three-dimensional meshwork, wherein the RNA oligomers are transcribed from a DNA template by RNA polymerase and fold and self-assemble simultaneously with transcription. The RNA origami tile has a structure including an internal kissing loop and an external kissing loop. The internal kissing loop stabilizes the structure within the tile, and the external kissing loop mediates the interaction between tiles to form a larger assembly. The internal kissing loop is a special tertiary structure formed between double-stranded RNA regions and is usually composed of an 8-base pair stem and a 4-nucleotide loop. The external kissing loop is located at the corner of the tile and is usually composed of a 6-base pair stem and a 4-nucleotide loop. These kissing loops play important roles in the folding and self-organization of RNA. The basic structure of the RNA origami tile is a structure (3H) including three helices, and each helix consists of a double-stranded RNA region. These helices are connected by an internal kissing loop. The angle (α) between the helices is determined by the arrangement of the internal kissing loop and is usually set at α = 120°. Due to this angle, six tiles gather to form a cylinder, resulting in a nanotube with a diameter of about 11 nm. The RNA origami tile incorporates a functional aptamer containing a growth factor-binding aptamer, a cell adhesion motif, and / or an enzymatically degradable sequence. Specifically, aptamers that bind to specific growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP), insulin-like growth factor (IGF), etc., aptamers that mimic cell adhesion motifs such as RGD (Arg-Gly-Asp) peptide, IKVAV (Ile-Lys-Val-Ala-Val) peptide, YIGSR (Tyr-Ile-Gly-Ser-Arg) peptide, etc., and / or aptamers containing sequences that can be degraded by specific enzymes such as matrix metalloproteinases (MMP-1, MMP-2, MMP-9, etc.), cathepsin, elastase, etc. can be incorporated. These functional aptamers are linked to the RNA origami tiles via linker sequences (e.g., AAA, AAAA, UUUU, etc.). The length and composition of the linker sequence can be adjusted to optimize the function of the aptamer. For example, a longer linker (e.g., AAAAAA) may provide greater structural freedom to the aptamer and potentially improve the binding efficiency to the target molecule. On the other hand, a shorter linker (e.g., AA) can more precisely control the position of the aptamer. The mechanical properties of the RNA origami tiles can be adjusted by modifying the RNA sequence. Specifically, sequences with a high GC content form a more rigid structure, while sequences with a high AU content form a more flexible structure. Since GC base pairs form three hydrogen bonds, while AU base pairs form only two hydrogen bonds, the higher the GC content, the more the structure is stabilized and the more rigid it becomes. For example, by reducing the GC content in the helix region from 75% to 50%, the rigidity of the nanotube can be significantly decreased. Also, by changing structural features such as the length and position of double-stranded regions, the number and arrangement of kissing loops, etc., the persistence length of the nanotube can be adjusted in the range of 0.8 μm to 3.4 μm. The persistence length is an indicator representing the rigidity of the polymer and is the characteristic length at which the directional correlation due to thermal fluctuations is lost. The longer the persistence length, the more rigid the polymer and the more likely it is to maintain a linear structure. For example, increasing the length of the helix can increase the persistence length, and introducing double-stranded overhangs can decrease the persistence length. Furthermore, by introducing specific sequence mutations, it is also possible to design RNA origami tiles that form a ring structure with a diameter of approximately 47 nm instead of a nanotube. This is achieved by reducing the GC content in the stem-loop region on the left side of the central helix from 75% to 50%. This mutation reduces the stability of the stem-loop and changes the angle within the tile, resulting in the formation of a circular structure instead of a linear nanotube.

[0018] A second aspect of the present invention is a biocompatible hydrogel comprising a DNA template, an RNA polymerase, nucleotide precursors, and magnesium ions, wherein RNA transcribed from the DNA template after injection or transplantation folds to form RNA origami tiles, and further self-organizes to form a three-dimensional extracellular matrix mimicking structure. The DNA template is a double-stranded DNA encoding the sequence of the RNA origami tile and contains an appropriate promoter sequence such as a T7 promoter, an SP6 promoter, or a T3 promoter. The concentration of the DNA template is usually in the range of 4-8 ng / μL. The DNA template can be prepared by PCR amplification, chemical synthesis, or recombinant DNA technology. The RNA polymerase is an enzyme that transcribes RNA from the DNA template, and bacteriophage-derived RNA polymerases such as T7 RNA polymerase, SP6 RNA polymerase, or T3 RNA polymerase are preferred. The concentration of the RNA polymerase is usually in the range of 0.2-1 U / μL. The nucleotide precursors are a mixture of ATP, GTP, CTP, and UTP, and the concentration of each nucleotide is usually in the range of 1-4 mM. These nucleotides are the basic components necessary for RNA synthesis. The magnesium ions are ions necessary for the activity of the RNA polymerase and also play an important role in the folding and stabilization of RNA. The concentration of the magnesium ions is usually in the range of 6-20 mM. The concentration of the magnesium ions has an important influence on the formation and stability of the RNA origami. At low concentrations (1 mM or less), transcription proceeds, but the accurate folding of the RNA origami is inhibited. At an appropriate concentration (about 6 mM), the RNA origami is accurately folded to form stable nanotubes. At high concentrations (20 mM or more), especially in a flexible dsOV design, the formation of nanotube bundles is promoted. The hydrogel is composed of a biocompatible polymer selected from hyaluronic acid, alginic acid, polyethylene glycol (PEG), collagen, fibrin, chitosan, agarose, polyvinyl alcohol (PVA), or a combination thereof. The concentration of the hydrogel is usually in the range of 1-5% (w / v). The selection of the hydrogel is based on factors such as the characteristics of the target tissue, the required mechanical properties, and the degradation rate. The DNA template encodes a plurality of RNA oligomites with different promoters that respond to external stimuli. Specifically, the following promoter systems can be used: 1. Light-responsive promoter: A promoter that is activated in response to light of a specific wavelength. For example, the EL222 system that responds to blue light (450-490 nm), the PhyB-PIF6 system that responds to red light (650-670 nm), etc. 2. Temperature-responsive promoter: A promoter that is activated within a specific temperature range. For example, a heat shock promoter (such as the HSP70 promoter) that is activated at 42°C, a low-temperature-responsive promoter that is activated at low temperatures (32°C or below), etc. 3. pH-responsive promoter: A promoter that is activated within a specific pH range. For example, a promoter that is activated in an acidic environment (pH 6.5 or below), a promoter that is activated in an alkaline environment (pH 7.5 or above), etc. 4. Small molecule-responsive promoter: A promoter that is activated in response to a specific small molecule (such as a drug). For example, a tetracycline-responsive promoter (Tet system), an IPTG-responsive promoter (lac system), a rapamycin-responsive promoter (FRAP system), etc. 5. Oxygen concentration-responsive promoter: A promoter that is activated within a specific oxygen concentration range. For example, a HIF-1α-responsive promoter that is activated in a hypoxic environment (1-5% O2), etc. 6. Mechanical stimulus-responsive promoter: A promoter that is activated in response to a specific mechanical stimulus (such as stretching, compression, etc.). For example, the TIE2 promoter that responds to stretching stimuli, etc. By combining these promoter systems, a complex ECM-mimicking material that changes over time can be realized. For example, in the initial stage, nanotubes with a rigid WT design under the control of a constitutive promoter can be formed, and then, in response to external stimuli (such as light irradiation), nanotubes with a more flexible dsOV design can be designed to form. The magnesium ions are provided in a controlled release form. Specifically, the following methods can be used: 1. Microcapsules containing magnesium ions: Magnesium ions are encapsulated in microcapsules made of biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL), and are slowly released over time. 2. Liposomes containing magnesium ions: Magnesium ions are encapsulated in liposomes composed of lipid bilayers and are slowly released over time. 3. Compounds that form complexes with magnesium ions: Chelating agents such as ethylenediaminetetraacetic acid (EDTA) and citric acid form complexes with magnesium ions and release magnesium ions over time. 4. Magnesium ionophores: External magnesium ions are supplied using ionophores (e.g., A23187) that selectively permeate magnesium ions. The hydrogel can further contain a magnesium ionophore or a pore-forming protein. A magnesium ionophore is a molecule that selectively permeates magnesium ions and can control the formation of RNA origami by supplying magnesium ions from the outside. A pore-forming protein (e.g., α-hemolysin) forms pores that selectively permeate small molecules such as nucleotides, and can sustain the formation of RNA origami by supplying nucleotides from the outside. To control the formation of RNA origami within the hydrogel, the following two methods can be used: 1. Control Release of Magnesium Ions Using a Magnesium Ionophore: By supplying magnesium ions externally to a hydrogel containing a magnesium ionophore, the formation of RNA origami can be initiated. In this method, a hydrogel containing a DNA template, RNA polymerase, and nucleotide precursors is prepared and contains magnesium ions at a low concentration (1 mM or less). In this state, transcription proceeds, but the accurate folding of RNA origami is inhibited. After transplantation, when magnesium ions are supplied externally, the magnesium ions are taken into the hydrogel through the magnesium ionophore, and the formation of RNA origami is initiated. 2. Supply of Nucleotides Using a Pore-Forming Protein Such as α-Hemolysin: By supplying nucleotides externally to a hydrogel containing a pore-forming protein, the formation of RNA origami can be initiated and sustained. In this method, a hydrogel containing a DNA template, RNA polymerase, magnesium ions, and a pore-forming protein is prepared. In this state, since there are no nucleotide precursors, transcription does not proceed. After transplantation, when nucleotide precursors are supplied externally, the nucleotides are taken into the hydrogel through the pore-forming protein, and the formation of RNA origami is initiated. By continuing the supply of nucleotides, the formation of RNA origami can be sustained.

[0019] A third aspect of the present invention is a method for forming a tissue engineering scaffold using the above hydrogel for forming the above RNA origami-based extracellular matrix mimetic material. The method includes the following steps: 1. Prepare a biocompatible hydrogel containing a DNA template, RNA polymerase, nucleotide precursors, and magnesium ions. 2. Inject or transplant the hydrogel into the target site. 3. Optionally, apply an external stimulus (such as light, temperature change, pH change, administration of small molecules, etc.) to induce the expression of specific RNA origami tiles. 4. Permit the RNA oligomitter to self-assemble to form a three-dimensional extracellular matrix mimicking structure. The method is used for forming scaffolds for wound healing, cartilage regeneration, bone regeneration, nerve regeneration, promoting angiogenesis, or skin regeneration. By designing RNA oligomitters with mechanical and biochemical properties suitable for each tissue type, tissue-specific scaffolds can be formed.

Advantages of the Invention

[0020] The present invention provides the following advantages: 1. Realization of a dynamic ECM-mimicking material formed in situ after injection or transplantation: The RNA oligo-based ECM-mimicking material of the present invention is formed simultaneously with the transcription of RNA from a DNA template, so it does not require prefabrication and can form a three-dimensional network in situ after injection or transplantation. This provides a material that can conform to complex anatomical structures. Different from conventional prefabricated scaffolds, the material of the present invention can be injected in a liquid state and can perfectly conform to tissue defect sites with complex shapes. Also, since the formation process proceeds under physiological conditions (37°C, neutral pH), the risk of cytotoxicity is minimized. 2. Precise control of mechanical properties by sequence design: By designing the sequences of RNA oligomitters, the mechanical properties (persistence length 0.8 to 3.4 μm) of the resulting nanotubes and networks can be precisely controlled. This makes it possible to design ECM-mimicking materials with rigidities suitable for different tissue types, such as cartilage, skin, and nerve tissues. For example, nanotubes with a highly rigid WT design (persistence length about 3.4 μm) can be used for bone and cartilage tissues, nanotubes with a moderately rigid iSpi design (persistence length about 1.3 μm) can be used for skin and muscle tissues, and nanotubes with a flexible dsOV design (persistence length about 0.8 μm) can be used for nerve and adipose tissues. Also, by introducing specific sequence mutations, it is possible to design RNA oligomitters that form ring structures instead of nanotubes, which is useful for the formation of vascular-like structures. 3. Spatially and temporally controlled matrix properties that evolve in response to external stimuli: By using multiple DNA templates with different promoters, it is possible to realize an ECM-mimicking material whose properties change over time in response to external stimuli (such as light, temperature, pH, specific small molecules, etc.). This makes it possible to provide an optimal environment according to the developmental stage of the tissue. For example, during the wound healing process, nanotubes with high rigidity can be formed in the initial inflammatory phase to provide structural support, then nanotubes with growth factor-binding aptamers can be formed in the proliferation phase to promote cell proliferation, and finally nanotubes with cell adhesion motifs can be formed in the final remodeling phase to promote cell settlement. 4. Embedded scaffold remodeling mechanism by natural RNA degradation: RNA is naturally biodegradable and is degraded by extracellular ribonucleases. By utilizing this property, it is possible to realize a process in which the scaffold is gradually degraded in response to tissue growth and replaced by a new natural ECM. Also, the degradation rate can be adjusted by modifying the RNA sequence (such as 2'-O-methyl, 2'-fluoro, etc.). For example, in bone regeneration where long-term structural support is required, highly modified RNA can be used to slow down the degradation rate, and in skin regeneration where more rapid tissue regeneration is desired, RNA with less modification can be used to speed up the degradation rate. 5. Simple system requiring only minimal components (DNA template, RNA polymerase, nucleotides): The system of the present invention requires only minimal components, namely a DNA template, RNA polymerase, nucleotide precursors, and magnesium ions, and does not require a complex protein expression system or a large number of enzymes. This simplifies the manufacturing process and improves reproducibility. Also, all of these components are commercially available and can be manufactured in compliance with GMP (Good Manufacturing Practice). 6. Incorporation of functional aptamers for biological activity: Incorporation of growth factor-binding aptamers, cell adhesion motifs, and / or enzymatically degradable sequences into RNA origami tiles can provide bioactive ECM-mimetic materials that promote cell adhesion, proliferation, and differentiation. These functional aptamers can be precisely positioned at specific locations on the RNA origami tiles, and their density and distribution can be controlled. For example, incorporation of VEGF-binding aptamers can promote angiogenesis, incorporation of RGD-mimetic aptamers can promote cell adhesion, and incorporation of MMP-responsive sequences can promote cell invasion. 7. Reduced immunogenicity: The RNA origami-based ECM-mimicking materials of the present invention are composed of natural nucleotides, and therefore have lower immunogenicity compared to naturally derived materials containing heterologous proteins. In addition, RNA modifications (2'-O-methyl, 2'-fluoro, etc.) can minimize the activation of RNA recognition receptors (TLR3, TLR7, TLR8, etc.) that may induce innate immune responses. 8. Scalability and reproducibility: The system of the present invention is based on well-established methods for synthesis and amplification of DNA templates, production of RNA polymerase, and synthesis of nucleotide precursors, making it easy to scale up. In addition, the synthesis process is well-defined, minimizing batch-to-batch variation and ensuring high reproducibility. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Hereinafter, an embodiment of the present invention will be described in detail. The basic building blocks of the present invention are RNA origami tiles with specific sequences that are designed to fold during transcription and then self-assemble into three-dimensional networks. RNA origami tiles are designed to have the following characteristics: - Ability to self-organize into 3D mesh-like networks with controllable pore size - Incorporation of multiple functional aptamers (growth factor binding, presentation of cell adhesion motifs, enzymatically degradable sequences) - Ability to adjust mechanical properties (rigidity or flexibility) by sequence modification The basic structure of the RNA origami tile is a structure containing three helices (3H), and each helix consists of a double-stranded RNA region. These helices are linked by internal kissing loops. The internal kissing loop is a special tertiary structure formed between double-stranded RNA regions and plays a role in stabilizing the structure within the tile. The structure of the internal kissing loop usually consists of an 8-base pair stem and a 4-nucleotide loop. The stem region is composed of a sequence with a high GC content (usually 75% or more), providing high stability. The loop region is usually composed of a GNRA tetraloop (G-N-R-A, where N is any nucleotide and R is a purine base), which is known to form a particularly stable structure. The arrangement of the internal kissing loops determines the angle (α) between the helices, which is an important factor determining the overall shape of the tile. The external kissing loop plays a role in mediating the interaction between tiles to form a larger assembly. Specifically, the external kissing loops located at the corners of the tiles interact with complementary external kissing loops of other tiles, linking the tiles together. Due to the geometric arrangement of this linkage, the tiles self-organize to form nanotubes. The structure of the external kissing loop is usually composed of a 6-base pair stem and a 4-nucleotide loop. The stem region is composed of sequences with a medium GC content (usually 50-60%), providing a balance of moderate stability and flexibility. The loop region is usually composed of a UUCG tetraloop, which is known to form a particularly stable structure. The sequence of the external kissing loop is designed so that the binding energy with the complementary kissing loop is -8 kcal / mol or less. Also, by designing to minimize the difference in binding energy between different kissing loop pairs, uniform nanotube formation is promoted. The diameter of the nanotube is determined by the angle (α) between the helices within the tile. When α = 120°, six tiles gather to form a cylindrical shape, resulting in a nanotube with a diameter of approximately 11 nm. This angle can be adjusted by the arrangement and length of the internal kissing loops. For example, the angle can be increased by shortening the distance between the internal kissing loops and decreased by lengthening the distance. The mechanical properties of the RNA origami tile can be adjusted by the following factors: 1. GC content: Since GC base pairs form stronger hydrogen bonds than AU base pairs, sequences with a high GC content form a more rigid structure. Specifically, the rigidity of the nanotube can be adjusted by changing the GC content in the helix region. For example, it has been confirmed that a sequence with a GC content of 75% exhibits approximately 1.5 times the rigidity compared to a sequence with a GC content of 50%. In particular, the GC content in the stem-loop region on the left side of the central helix has a great influence on the shape (linear or circular) of the nanotube. When the GC content in this region is decreased from 75% to 50%, the stability of the stem-loop decreases, the angle within the tile changes, and a circular structure is formed instead of a linear nanotube. 2. Length of the helix: The longer the helix, the greater the rigidity of the nanotube. Specifically, by increasing the length of the helix, the persistence length of the nanotube can be increased. For example, it has been confirmed that when the length of the helix is increased from 10 base pairs to 15 base pairs, the persistence length becomes approximately 1.5 times. However, if the helix is too long, the folding efficiency of the tile may decrease, so it is usually set in the range of 10 - 20 base pairs. 3. Number and arrangement of kissing loops: The greater the number of kissing loops, the more stable the structure and the greater the rigidity. Specifically, by increasing the number of internal kissing loops, the structure within the tile is stabilized and the rigidity of the nanotube is increased. Also, by increasing the number of external kissing loops, the interaction between tiles is strengthened and the stability of the nanotube is improved. For example, in a standard design, each tile has 2 external kissing loops, but it has been confirmed that increasing this to 3 doubles the stability of the nanotube. 4. Presence or absence of double-stranded overhang: By introducing a double-stranded overhang, the flexibility of the nanotube can be increased. Specifically, by introducing a double-stranded overhang (usually 10 - 20 base pairs in length) at a specific position of the tile, the persistence length of the nanotube can be decreased. For example, in the dsOV design where a double-stranded overhang is introduced into the standard WT design (persistence length of about 3.4 μm), it has been confirmed that the persistence length decreases to about 0.8 μm. The double-stranded overhang is connected to the tile via a three-way junction, and the position and structure of this junction also affect the flexibility of the nanotube. In the present invention, the following three basic RNA origami tile designs are provided: 1. WT (Wild Type): A basic design that forms highly rigid nanotubes with a persistence length of approximately 3.4 μm. This design has a high GC content (about 75% in the helix region) and no double-stranded overhangs. The nanotubes of the WT design are suitable for the regeneration of tissues that require high mechanical strength, such as bone and cartilage tissues. Also, the nanotubes of the WT design have low bundling ability even under high magnesium concentrations and tend to exist as individual nanotubes. 2. iSpi: A design incorporating the iSpinach aptamer. It forms nanotubes with moderate rigidity and a persistence length of approximately 1.3 μm. The iSpinach aptamer is an RNA sequence that binds to fluorescent dyes such as DFHBI-1T and emits fluorescence, and can be used for fluorescence imaging. The nanotubes of the iSpi design are suitable for the regeneration of tissues that require moderate mechanical strength, such as skin and muscle tissues. The iSpinach aptamer is usually linked to the 3' end of the tile via a 2-uracil linker. 3. dsOV: A design with double-stranded overhangs. It forms flexible nanotubes with a persistence length of approximately 0.8 μm and shows bundling ability under high magnesium concentrations. The nanotubes of the dsOV design are suitable for the regeneration of tissues where flexibility is important, such as nerve and adipose tissues. The double-stranded overhangs are connected to the tile via a three-way branch point, which is usually located near the 3' end of the tile but not at the 3' end itself. The length of the double-stranded overhangs is usually 15 - 20 base pairs, and its sequence is set to have a GC content of approximately 50%. Furthermore, by introducing specific sequence mutations, it is possible to design RNA origami tiles that form ring structures with a diameter of approximately 47 nm instead of nanotubes. This is achieved by reducing the GC content in the stem-loop region on the left side of the central helix from 75% to 50%. This mutation decreases the stability of the stem-loop, changes the angle within the tile, and forms a circular structure instead of a linear nanotube. Specifically, this mutation increases the angle (β) between the double strands containing the upper left and lower left kissing loops from approximately 50° in the WT design to approximately 90° in the WT-mut design. This change in angle enhances the tendency of the tiles to arrange in a circular pattern. RNA origami tiles that form ring structures are useful for the formation of vascular-like structures and the mimicking of circular cytoskeletal structures. Functional aptamers can be incorporated at specific positions in RNA origami tiles. Specifically, the following aptamers can be incorporated: 1. Growth factor-binding aptamers: Aptamers that bind to specific growth factors such as VEGF, bFGF, PDGF, TGF-β, BMP, IGF. These aptamers are designed to have high affinity (usually Kd = 1 - 100 nM) and specificity for the target growth factor. Growth factor-binding aptamers can induce specific cellular responses such as cell proliferation, differentiation, and migration by binding to the growth factor and releasing it gradually. For example, incorporating a VEGF-binding aptamer can promote angiogenesis, and incorporating a TGF-β-binding aptamer can promote chondrocyte differentiation. 2. Cell adhesion motif aptamers: Aptamers that mimic cell adhesion motifs such as the RGD peptide, IKVAV peptide, YIGSR peptide. These aptamers interact with receptors such as integrins on the cell surface and promote cell adhesion. For example, the RGD-mimicking aptamer interacts with integrin αvβ3 and promotes the adhesion of fibroblasts and endothelial cells. The IKVAV-mimicking aptamer interacts with the 67 kDa laminin receptor and promotes neuronal adhesion and neurite outgrowth. The YIGSR-mimicking aptamer interacts with the 67 kDa laminin receptor and promotes epithelial cell adhesion. 3. Biotin Aptamer: An aptamer that enables binding to a biotinylated lipid membrane. The biotin aptamer has a high affinity for biotin (Kd = 5.7 μM) and enables interaction with a membrane containing biotinylated lipids. This allows RNA origami nanotubes to form a cortex-like structure along the membrane. The biotin aptamer is typically about 80 nucleotides in length and is linked to the tile via an AAA (3 adenine) linker. 4. Enzymatically Cleavable Sequences: Aptamers containing sequences that can be cleaved by specific enzymes such as MMP-1, MMP-2, MMP-9, etc. These sequences contain cleavage sites for specific enzymes and enable the realization of ECM-mimicking materials that are degraded in response to enzymes secreted by cells. For example, by incorporating an RNA sequence that mimics the MMP-2 / 9 cleavage site (PLGLAG), nanotubes that are degraded in response to MMP-2 / 9 activated during angiogenesis and wound healing processes can be designed. This promotes cell infiltration and tissue remodeling. These aptamers are linked to RNA origami tiles via a linker sequence (e.g., AAA, AAAA, UUUU, etc.). The length and composition of the linker sequence can be adjusted to optimize the function of the aptamer. For example, a longer linker (e.g., AAAAAA) may provide greater structural freedom to the aptamer and potentially improve the binding efficiency to the target molecule. On the other hand, a shorter linker (e.g., AA) can more precisely control the position of the aptamer. The arrangement density of the aptamers is also an important design parameter. A high-density aptamer arrangement may exhibit a stronger biological effect but may also inhibit the folding and self-assembly of the RNA origami. Generally, it is recommended to incorporate 1 - 3 aptamers into each tile. It is also possible to incorporate different types of aptamers into the same tile, enabling the design of nanotubes with multiple biological functions.

[0022] The design process of RNA origami tiles is carried out in the following steps: 1. Design of the basic structure: Determine the basic structure such as the number and length of helices, the arrangement of internal and external kissing loops, and the presence or absence of double-stranded overhangs. 2. Sequence optimization: Optimize the sequence considering factors such as GC content, stability of secondary structure, and binding energy of kissing loops. Software tools such as Revolvr, RNAfold, and Mfold are used for sequence optimization. 3. Incorporation of functional aptamers: Select functional aptamers according to the purpose and incorporate them into the tiles via appropriate linker sequences. Analyze the overall structure with tools such as RNAfold to ensure that the secondary structure of the aptamer is correctly formed. 4. Molecular dynamics simulation: Use coarse-grained molecular dynamics simulation tools such as oxRNA to simulate the folding and self-assembly of the designed RNA origami tiles and verify the validity of the design. 5. Experimental verification: Synthesize the DNA template encoding the designed RNA origami tiles and verify the formation and self-assembly of RNA oligomers through in vitro transcription experiments. The formed nanotubes can be observed by atomic force microscopy (AFM), transmission electron microscopy (TEM), or fluorescence microscopy (when the iSpinach aptamer is incorporated). The ECM-mimicking material of the present invention, different from conventional biomaterials, does not require prefabrication and is formed in situ. This system is composed of a biocompatible hydrogel containing the following components: 1. DNA template encoding RNA origamitiles: A double-stranded DNA template encoding the sequence of RNA origamitiles. It contains appropriate promoter sequences such as T7 promoter, SP6 promoter, T3 promoter, etc. The concentration of the DNA template is usually in the range of 4 - 8 ng / μL. The DNA template can be prepared by PCR amplification, chemical synthesis, or recombinant DNA technology. The design of the template is carried out considering the following elements: - Promoter sequence: A promoter sequence that determines the starting point of RNA transcription. The T7 promoter is most commonly used, but SP6 promoter, T3 promoter, etc. can also be used. - Transcription start sequence: A sequence to enhance the efficiency and accuracy of transcription. Usually, a sequence with a high guanine content such as GGAA is used. - RNA origamitile sequence: The sequence of the designed RNA origamitiles. - Transcription termination sequence: A sequence to promote the termination of transcription. Usually, multiple thymidine residues (e.g., TTTTTT) are used. When using a promoter that responds to external stimuli, the following designs are possible: - Light-responsive promoter: EL222 system (blue light response), PhyB-PIF6 system (red light response), etc. - Temperature-responsive promoter: HSP70 promoter (heat shock response), etc. - pH-responsive promoter: A promoter that is activated in an acidic environment, etc. - Small molecule-responsive promoter: Tet system (tetracycline response), lac system (IPTG response), etc. 2. RNA polymerase: An enzyme that transcribes RNA from a DNA template. RNA polymerases derived from bacteriophages such as T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase are preferred. The concentration of RNA polymerase is usually in the range of 0.2 - 1 U / μL. The selection of RNA polymerase needs to match the promoter sequence to be used. For example, when using the T7 promoter, T7 RNA polymerase needs to be used. The activity of RNA polymerase depends on conditions such as temperature, pH, and ionic strength. Optimal activity is usually obtained under the conditions of 37°C, pH 7.5 - 8.0, 40 - 50 mM NaCl or KCl, and 6 - 20 mM MgCl2. 3. Nucleotide precursors: A mixture of ATP, GTP, CTP, and UTP. The concentration of each nucleotide usually ranges from 1 - 4 mM. These nucleotides are the basic components necessary for RNA synthesis. The concentration of nucleotide precursors affects the rate and duration of RNA synthesis. At high concentrations (above 4 mM), the synthesis rate is faster, but the accumulation of pyrophosphate produced as a by - product may deplete magnesium ions. At low concentrations (below 1 mM), the synthesis rate is slower but persists for a longer time. The use of modified nucleotides (such as 2'-O - methyl - NTP, 2'-fluoro - NTP, etc.) can improve the stability of the generated RNA. These modified nucleotides increase resistance to degradation by ribonucleases. However, the use of modified nucleotides may affect RNA folding and self - organization, so prior verification is required. 4. Magnesium ions: Ions necessary for the activity of RNA polymerase and also play an important role in RNA folding and stabilization. The concentration of magnesium ions usually ranges from 6 - 20 mM. The concentration of magnesium ions has an important impact on the formation and stability of RNA origami. At low concentrations (below 1 mM), transcription proceeds, but the accurate folding of RNA origami is inhibited. At an appropriate concentration (about 6 mM), RNA origami is accurately folded to form stable nanotubes. At high concentrations (above 20 mM), especially in flexible dsOV designs, the formation of nanotube bundles is promoted. The method of supplying magnesium ions is an important factor for controlling the formation of RNA origami. The following methods can be used: - Direct addition: Magnesium ions are directly added during the preparation of the hydrogel. - Supply via a magnesium ionophore: Magnesium ions are supplied externally to a hydrogel containing a magnesium ionophore. - Controlled release system: It is slowly released over time using microcapsules or liposomes containing magnesium ions. 5. Biocompatible polymers: Polymers for forming hydrogels containing the above components. Selected from hyaluronic acid, alginic acid, PEG, collagen, fibrin, chitosan, agarose, PVA, or combinations thereof. The concentration of the hydrogel is usually in the range of 1-5% (w / v). The selection of the hydrogel is based on factors such as the characteristics of the target tissue, required mechanical properties, degradation rate, etc. For example: - Hyaluronic acid: Suitable for tissues such as skin and cartilage. It is a natural ECM component and has high biocompatibility. - Alginic acid: It has the property of cross-linking with calcium ions and rapidly gelling. Suitable for cell encapsulation. - PEG: A synthetic polymer, and its mechanical properties and degradation rate can be precisely controlled. Applicable to various tissues. - Collagen: Suitable for tissues such as bone, skin, and tendon. It is a natural ECM component and contains cell adhesion sites. - Fibrin: Suitable for wound healing, angiogenesis, etc. It rapidly gels by the blood coagulation cascade. The physical properties of the hydrogel (such as viscosity, gelation time, mechanical strength, etc.) can be adjusted by factors such as the type and concentration of the polymer, the type and concentration of the cross-linking agent, pH, temperature, etc. After injection or transplantation, these components are activated and the following processes proceed: 1. RNA transcription from a DNA template: RNA polymerase synthesizes RNA from a DNA template. Transcription starts from a promoter sequence and proceeds towards the 3' end of the template. The transcription rate is usually 20 - 50 nucleotides / second. For example, the synthesis of a 500-nucleotide RNA origami tile takes about 10 - 25 seconds. 2. Folding of RNA origami tiles during transcription: The synthesized RNA folds during transcription to form RNA origami tiles. The folding of RNA proceeds stepwise as transcription progresses. First, local secondary structures (such as stem-loops) are formed, and then long-range tertiary structures (such as internal kissing loops) are formed. The folding rate depends on factors such as the complexity of the RNA sequence, GC content, temperature, and ionic strength. 3. Formation of nanotubes through interactions between tiles: The formed tiles interact via external kissing loops to form nanotubes. The interaction between tiles depends on factors such as the sequence and structure of the external kissing loops, magnesium ion concentration, and temperature. The formation rate of nanotubes depends on the tile concentration and usually proceeds on a time scale of several minutes to several hours. 4. Formation of a 3D network through self-organization of nanotubes: The formed nanotubes further interact to form a 3D mesh-like network. The formation of the network depends on factors such as the concentration, length, rigidity, and surface properties of the nanotubes. The formation rate of the network usually proceeds on a time scale of several hours to several days. This process proceeds under isothermal conditions (37°C) and does not require non-physiological conditions such as heat annealing. Also, since the synthesis and folding of RNA proceed continuously, the formation of the material starts immediately after injection or implantation and progresses over time. To control the formation of RNA origami within a hydrogel, the following two methods can be used: 1. Controlled Release of Magnesium Ions Using a Magnesium Ionophore: By supplying magnesium ions externally to a hydrogel containing a magnesium ionophore, the formation of RNA origami can be initiated. In this method, a hydrogel containing a DNA template, RNA polymerase, and nucleotide precursors is prepared and contains magnesium ions at a low concentration (1 mM or less). In this state, transcription proceeds, but the accurate folding of RNA origami is inhibited. After transplantation, when magnesium ions are supplied externally, the magnesium ions are taken into the hydrogel through the magnesium ionophore, and the formation of RNA origami is initiated. A magnesium ionophore is a molecule that selectively permeates magnesium ions. A typical magnesium ionophore is A23187 (calcimycin). A23187 has a high affinity for divalent cations (especially Ca2+ and Mg2+) and can transport these ions across the lipid bilayer. The concentration of A23187 is usually in the range of 10 - 50 μM. The advantage of this method is that the formation of RNA origami can be controlled externally. For example, by supplying magnesium ions at a specific timing after transplantation, the formation of RNA origami can be initiated. Also, by supplying magnesium ions stepwise, it is possible to sequentially form different types of RNA origami. 2. Supply of nucleotides using pore-forming proteins such as α-hemolysin: By supplying nucleotides from the outside to a hydrogel containing a pore-forming protein, the formation of RNA origami can be initiated and sustained. In this method, a hydrogel containing a DNA template, RNA polymerase, magnesium ions, and a pore-forming protein is prepared. In this state, since there are no nucleotide precursors, transcription does not proceed. After transplantation, when nucleotide precursors are supplied from the outside, nucleotides are taken into the hydrogel through the pore-forming protein, and the formation of RNA origami is initiated. By continuing the supply of nucleotides, the formation of RNA origami can be sustained.

[0023] α-Hemolysin is a pore-forming protein produced by Staphylococcus aureus and forms pores with a diameter of approximately 1.4 nm. These pores allow small molecules such as nucleotides (molecular weight <2 kDa) to pass through, but do not allow large molecules such as DNA templates and RNA polymerase to pass through. The concentration of α-hemolysin is usually in the range of 15 - 50 ng / μL. The advantage of this method is that the formation of RNA origami can be sustained over a long period of time. Even if the nucleotides in the hydrogel are consumed, the formation of RNA origami can be maintained by continuously supplying them from the outside. Also, since transcription by-products (such as pyrophosphate) are also discharged through the pores, the reaction environment is maintained. Furthermore, by sequentially supplying different types of nucleotides (such as natural nucleotides and modified nucleotides), it is also possible to form RNA origami with different properties. The RNA origami ECM of the present invention has the following programmable properties: **Mechanical tunability**: By modifying the RNA sequence, the stiffness of the resulting matrix can be precisely controlled to match specific tissue types (persistence length 0.8 to 3.4 μm). For example, sequences with a high GC content form a more rigid structure, while sequences with a high AU content form a more flexible structure. Additionally, the introduction of double-stranded overhangs can increase flexibility. The persistence length is an indicator of the polymer's stiffness and is the characteristic length at which the direction correlation due to thermal fluctuations is lost. The longer the persistence length, the more rigid the polymer and the more likely it is to maintain a linear structure. The persistence length of RNA origami nanotubes can be calculated using coordinates extracted from atomic force microscopy (AFM) images. Specifically, the square of the end-to-end distance of the nanotube is plotted against the contour length and fitted to a theoretical formula to obtain the persistence length. Specifically, RNA origami tiles with stiffness suitable for the following tissue types can be designed: 1. Bone and cartilage tissue: Rigid nanotubes with a WT design (persistence length of approximately 3.4 μm). The native ECM of bone tissue is mainly composed of collagen type I and hydroxyapatite and has high mechanical strength (elastic modulus of approximately 20 GPa). The native ECM of cartilage tissue is mainly composed of collagen type II and proteoglycans and has moderate mechanical strength (elastic modulus of approximately 0.5 - 1 MPa). Nanotubes with a WT design exhibit high stiffness due to their high GC content (approximately 75% in the helix region) and lack of double-stranded overhangs, making them suitable for the regeneration of these tissues. Additionally, incorporating a BMP-binding aptamer into the WT design nanotubes can promote bone formation. 2. Skin and muscle tissues: Nanotubes with moderate rigidity of the iSpi design (persistent length of approximately 1.3 μm). The natural ECM of skin tissue is mainly composed of collagen types I, III, and elastin, and has moderate mechanical strength (elastic modulus of approximately 0.1 - 0.2 MPa) and elasticity. The natural ECM of muscle tissue is mainly composed of collagen types I, III, IV, and laminin, and has moderate mechanical strength (elastic modulus of approximately 0.01 - 0.02 MPa) and contractility. The nanotubes of the iSpi design exhibit moderate rigidity due to the incorporation of the iSpinach aptamer and are suitable for the regeneration of these tissues. Additionally, by incorporating the bFGF-binding aptamer into the nanotubes of the iSpi design, the proliferation of fibroblasts can be promoted. 3. Nerve and adipose tissues: Flexible nanotubes of the dsOV design (persistent length of approximately 0.8 μm). The natural ECM of nerve tissue is mainly composed of collagen type IV, laminin, and proteoglycan, and has low mechanical strength (elastic modulus of approximately 0.001 - 0.01 MPa) and high flexibility. The natural ECM of adipose tissue is mainly composed of collagen types I, IV, and laminin, and has very low mechanical strength (elastic modulus of approximately 0.0001 - 0.001 MPa) and high flexibility. The nanotubes of the dsOV design exhibit high flexibility due to the introduction of double-stranded overhangs and are suitable for the regeneration of these tissues. Additionally, by incorporating the NGF-binding aptamer into the nanotubes of the dsOV design, the survival of nerve cells and neurite outgrowth can be promoted. 4. Vascular tissue: Ring structure of the WT-mut design. The natural ECM of vascular tissue is mainly composed of collagen types I, III, elastin, and fibronectin, and has a layered structure and a circumferential orientation. The ring structure of the WT-mut design is formed by reducing the GC content in the stem-loop region on the left side of the central helix and forms a circular structure with a diameter of approximately 47 nm. This mimics the basic structure of blood vessels and is suitable for the regeneration of vascular tissue. Additionally, by incorporating the VEGF-binding aptamer into the ring structure of the WT-mut design, the proliferation of vascular endothelial cells and angiogenesis can be promoted. **Spatiotemporal control**: By including multiple DNA templates with different promoters that respond to external stimuli (light, temperature, pH, small molecules), it becomes possible for the matrix properties to evolve over time. Specifically, the following promoter systems can be used: 1. Light-responsive promoter: A promoter that is activated in response to light of a specific wavelength. For example, the EL222 system that responds to blue light (450 - 490 nm), the PhyB-PIF6 system that responds to red light (650 - 670 nm), etc. The EL222 system consists of the blue light receptor EL222 and its target promoter. When EL222 absorbs blue light, it undergoes a structural change, binds to DNA, and activates the promoter. When light irradiation stops, EL222 returns to its original state and the activation of the promoter ceases. This system can precisely control gene expression levels according to the intensity and irradiation time of light. The advantage of the light-responsive promoter is that it can control gene expression non-invasively with high spatiotemporal precision. For example, by irradiating a specific region with light of a specific wavelength, the formation of RNA origami can be induced only in that region. Also, by using multiple promoters that respond to different wavelengths of light, it is possible to selectively form different types of RNA origami. 2. Temperature-responsive promoter: A promoter that is activated within a specific temperature range. For example, a heat shock promoter (such as the HSP70 promoter) that is activated at 42°C, a cold-responsive promoter that is activated at low temperatures (32°C or lower), etc. The heat shock promoter contains a binding site for the heat shock factor (HSF). At normal temperatures, HSF forms a complex with chaperone proteins and cannot bind to DNA. When the temperature rises, the chaperone protein binds to the denatured protein, so HSF is released, binds to the promoter, and activates gene expression. The advantage of a temperature-responsive promoter is that gene expression can be controlled by local temperature changes (e.g., ultrasonic irradiation, magnetic hyperthermia, etc.). For example, by heating a specific region, the formation of RNA origami can be induced only in that region. Also, by using multiple promoters with different temperature thresholds, it is possible to form different types of RNA origami stepwise as the temperature rises. 3. pH-responsive promoter: A promoter that is activated in a specific pH range. For example, a promoter that is activated in an acidic environment (pH 6.5 or lower), a promoter that is activated in an alkaline environment (pH 7.5 or higher), etc. A pH-responsive promoter contains a binding site for a pH-dependent DNA-binding protein. For example, a promoter that is activated in an acidic environment contains a binding site for a transcription factor that binds to DNA at acidic pH. The advantage of a pH-responsive promoter is that gene expression can be controlled in response to physiological or pathological pH changes in tissues. For example, in pathological conditions such as inflammation and tumors, it is known that the pH of tissues decreases. By using a promoter that is activated in an acidic environment, the formation of RNA origami can be induced specifically in these pathological conditions. Also, in the wound healing process, it is known that the environment is acidic in the initial inflammatory phase and neutral in the later proliferation and remodeling phases. By using promoters with different pH responses, it is possible to form RNA origami suitable for each stage of wound healing. 4. Small molecule-responsive promoter: A promoter that is activated in response to a specific small molecule (such as a drug). For example, a tetracycline-responsive promoter (Tet system), an IPTG-responsive promoter (lac system), a rapamycin-responsive promoter (FRAP system), etc. The Tet system is composed of a promoter that is activated or suppressed in the presence of tetracycline (or doxycycline). In the TetON system, the promoter is activated in the presence of tetracycline, and in the TetOFF system, the promoter is suppressed in the presence of tetracycline. The advantage of small molecule-responsive promoters is that gene expression can be controlled by drug administration. For example, by administering a specific drug, the formation of RNA origami can be initiated or stopped. Also, by using multiple promoters that respond to different drugs, it is possible to selectively form different types of RNA origami. Furthermore, the gene expression level can be precisely controlled by the dosage and timing of drug administration. 5. Oxygen concentration-responsive promoter: A promoter that is activated within a specific oxygen concentration range. For example, the HIF-1α-responsive promoter that is activated in a hypoxic environment (1-5% O2). The HIF-1α-responsive promoter contains a binding site for the hypoxia-inducible factor (HIF-1α). Under normal oxygen concentrations, HIF-1α is hydroxylated by prolyl hydroxylase (PHD), ubiquitinated, and degraded. In a hypoxic environment, the activity of PHD decreases, HIF-1α is stabilized, binds to the promoter, and activates gene expression. The advantage of oxygen concentration-responsive promoters is that gene expression can be controlled in response to physiological or pathological changes in tissue oxygen concentration. For example, in a hypoxic environment such as ischemic tissue or inside a tumor, the HIF-1α-responsive promoter is activated, inducing the formation of RNA origami. This makes it possible to exert a therapeutic effect specific to the hypoxic environment. Also, in tissue engineering approaches, the center of large tissue constructs is prone to hypoxia. By using the HIF-1α-responsive promoter, it is possible to form RNA origami specifically in the hypoxic region and promote angiogenesis. 6. Mechanosensitive promoter: A promoter that is activated in response to specific mechanical stimuli (stretching, compression, etc.). For example, the TIE2 promoter that responds to stretching stimuli. Mechanosensitive promoters contain binding sites for transcription factors (e.g., YAP / TAZ, MRTF-A / SRF, etc.) that are activated by mechanical stimuli. The advantage of a mechanically responsive promoter is that it can control gene expression in response to changes in the mechanical environment of tissues. For example, in bone tissue, it is known that mechanical loading promotes bone formation. By using a mechanically responsive promoter, it becomes possible to induce the formation of RNA origami specifically in regions subjected to mechanical loading and promote bone formation. Also, in mechanically active tissues such as myocardial tissue and vascular tissue, the formation of RNA origami can be adjusted according to the mechanical activity of the tissue.

[0024] By combining these promoter systems, it is possible to realize a complex ECM-mimicking material that changes over time. For example, in the initial stage, nanotubes with a rigid WT design under constitutive promoter control can be formed, and then, it can be designed to form more flexible dsOV-design nanotubes in response to external stimuli (such as light irradiation). As a specific application example, an ECM-mimicking material adapted to the wound healing process can be designed as follows: 1. Initial inflammatory phase (0 - 2 days): Form nanotubes with a rigid WT design under constitutive promoter control to provide structural support. 2. Proliferative phase (3 - 10 days): Form nanotubes with an iSpi design having a PDGF-binding aptamer under the control of a pH-responsive promoter (activated in an acidic environment) to promote fibroblast proliferation. 3. Remodeling phase (after 11 days): Form nanotubes with a dsOV design having an RGD-mimicking aptamer under the control of a temperature-responsive promoter (gradually activated at 37°C) to promote cell adhesion and tissue remodeling. **Biodegradability**: Natural RNA degradation provides a mechanism for incorporating scaffold remodeling, and the degradation rate can be adjusted by sequence modification and protective chemical modification. The degradation rate of RNA can be adjusted by the following methods: 1. 2'-O-Methyl modification: By modifying the oxygen atom at the 2'-position of RNA with a methyl group, the resistance to degradation by ribonucleases can be increased. 2'-O-Methyl modification changes the structure of RNA from the A-form to the B-form, inhibiting the recognition by ribonucleases. Also, 2'-O-methyl modification has the effect of improving the thermal stability of RNA. 2'-O-Methyl-NTPs are commercially available and can be used in standard in vitro transcription reactions. However, if all nucleotides are 2'-O-methyl modified, the activity of RNA polymerase may decrease. Therefore, usually only some nucleotides (e.g., only pyrimidine nucleotides) are modified. 2. 2'-Fluoro modification: By substituting the oxygen atom at the 2'-position of RNA with a fluorine atom, the resistance to degradation by ribonucleases can be increased. 2'-Fluoro modification maintains the structure of RNA in the A-form while reducing the nucleophilicity at the 2'-position and inhibiting hydrolysis by ribonucleases. 2'-Fluoro-NTPs are also commercially available and can be used in standard in vitro transcription reactions. 2'-Fluoro modification has less impact on the structure of RNA than 2'-O-methyl modification, so it has less effect on the folding and function of RNA. 3. Modification of the phosphate backbone: By introducing modifications such as phosphorothioate bonds, the resistance to degradation by nucleases can be increased. A phosphorothioate bond is one in which one of the oxygen atoms of the phosphate group is replaced by a sulfur atom, inhibiting recognition by nucleases. Since phosphorothioate modification is usually introduced post-transcriptionally, it needs to be chemically modified after in vitro transcription. 4. Co-encapsulation of ribonuclease inhibitors: By co-encapsulating ribonuclease inhibitors in hydrogels, the degradation of RNA can be delayed. Ribonuclease inhibitors include vanadium complexes, RNasin, SUPERase·In, etc. These inhibitors bind to the active site of ribonucleases and inhibit their activity. The concentration of ribonuclease inhibitors is usually in the range of 1 - 2 U / μL. These modifications can be introduced uniformly throughout the entire RNA origami or selectively into specific regions. For example, high protection can be designed for structurally important regions (such as internal kissing loops, external kissing loops, etc.), and low protection can be provided for regions where biodegradability is required (such as enzymatically degradable sequences). RNA origamis with degradation profiles suitable for different tissue types can be designed. For example: 1. Bone tissue: Since long-term structural support is required, highly modified RNA (containing both 2'-O-methyl and 2'-fluoro) is used to make the degradation rate very slow (half-life: weeks to months). 2. Cartilage tissue: Since moderate structural support is required, moderately modified RNA (containing either 2'-O-methyl or 2'-fluoro) is used to make the degradation rate moderate (half-life: 1 - 2 weeks). 3. Skin tissue: Since relatively rapid tissue regeneration is desired, lightly modified RNA (only some nucleotides are modified) is used to make the degradation rate relatively fast (half-life: days to 1 week). 4. Nerve tissue: Since long-term induction is required, highly modified RNA is used to make the degradation rate very slow (half-life: weeks to months). **Functionality**: Various biological functions can be realized by functional aptamers incorporated into RNA origami tiles. Specifically, the following functions can be realized: 1. Binding and slow release of growth factors: By incorporating a growth factor-binding aptamer, a specific growth factor can be bound and slowly released. For example, by incorporating an aptamer that binds to VEGF, angiogenesis can be promoted. Growth factor-binding aptamers are designed to have high affinity (usually Kd = 1-100 nM) and specificity for target growth factors. The binding of the aptamer to the growth factor is reversible, and the dynamic equilibrium of binding and dissociation enables the slow release of the growth factor. The release rate can be adjusted by the affinity of the aptamer, the density of the aptamer, and environmental conditions (such as pH, ionic strength, etc.). For example, RNA origami nanotubes incorporating a VEGF-binding aptamer (Kd = 20 nM) bind VEGF and release it gradually, promoting the proliferation, migration, and lumen formation of vascular endothelial cells and inducing angiogenesis. Similarly, RNA origami nanotubes incorporating a PDGF-binding aptamer (Kd = 50 nM) bind PDGF and release it gradually, promoting the proliferation and migration of fibroblasts and accelerating wound healing. It is also possible to incorporate multiple growth factor-binding aptamers into the same RNA origami tile, thereby enabling the coordinated action of multiple growth factors. For example, incorporating an aptamer that binds both VEGF and bFGF can induce more effective angiogenesis. 2. Promotion of cell adhesion: Incorporating aptamers that mimic cell adhesion motifs such as RGD peptides, IKVAV peptides, and YIGSR peptides can promote cell adhesion. Aptamers that mimic cell adhesion motifs interact with cell surface receptors (such as integrins) and promote cell adhesion. These aptamers form specific three-dimensional structures and mimic natural peptide motifs. For example, RGD-mimicking aptamers interact with integrin αvβ3, α5β1, etc., and promote the adhesion of fibroblasts, endothelial cells, osteoblasts, etc. IKVAV-mimicking aptamers interact with the 67 kDa laminin receptor and promote the adhesion and neurite outgrowth of neurons. YIGSR-mimicking aptamers interact with the 67 kDa laminin receptor and promote the adhesion of epithelial cells. The density and distribution of aptamers mimicking cell adhesion motifs affect the efficiency of cell adhesion and cell morphology. High-density aptamer arrangements promote strong cell adhesion but may inhibit cell migration. On the other hand, low-density aptamer arrangements promote cell migration but may reduce adhesion strength. Also, the distribution pattern of aptamers (uniform distribution, gradient distribution, patchy distribution, etc.) also affects cell behavior. 3. Enzyme-responsive degradation: By incorporating aptamers containing sequences that can be degraded by specific enzymes such as MMPs, it is possible to realize ECM-mimicking materials that are degraded in response to enzymes secreted by cells. Aptamers containing enzyme-degradable sequences contain cleavage sites for specific enzymes and are selectively degraded in the presence of those enzymes. This promotes cell invasion and tissue remodeling. For example, by incorporating an RNA sequence mimicking the MMP-2 / 9 cleavage site (PLGLAG), nanotubes that are degraded in response to MMP-2 / 9 activated during angiogenesis and wound healing can be designed. Similarly, by incorporating an RNA sequence mimicking the MMP-1 cleavage site (GPQGIAGQ), nanotubes that are degraded in response to MMP-1 involved in collagen degradation can be designed. The arrangement of enzyme-degradable sequences affects the degradation pattern of RNA origami nanotubes. For example, by arranging enzyme-degradable sequences at the connection sites between tiles, nanotubes can be designed to be degraded into short segments in the presence of enzymes. On the other hand, by arranging enzyme-degradable sequences in specific regions within the tiles, the structure of the tiles can be changed in the presence of enzymes, and the mechanical properties of the nanotubes can be changed. 4. Cortex formation: By incorporating biotin aptamers and interacting with biotinylated lipid membranes, cortex-like structures can be formed along the membrane. The biotin aptamer has a high affinity for biotin (Kd = 5.7 μM) and enables interaction with membranes containing biotinylated lipids. As a result, RNA origami nanotubes align along the membrane and form a cortex-like structure. The cortex-like structure has functions such as maintaining cell morphology, regulating the mechanical properties of the membrane, and localizing membrane proteins. For example, RNA origami nanotubes incorporating biotin aptamers can bind to artificial cell membranes (such as liposomes) containing biotinylated lipids and increase the rigidity of the membrane. In addition, RNA origami nanotubes incorporating both biotin aptamers and RGD-mimicking aptamers can bind to cell membranes and promote cell adhesion and cytoskeleton rearrangement. The density and distribution of biotin aptamers affect the efficiency and structure of cortex formation. A high-density aptamer arrangement promotes strong cortex formation but may reduce membrane fluidity. On the other hand, a low-density aptamer arrangement maintains membrane fluidity but may reduce cortex stability.

Example

[0025] As an example of the present invention, a wound healing patch can be configured as follows: A hydrogel patch containing a DNA template encoding the following: 1. Rigid WT RNA oligomitter for initial structural support (persistence length of about 3.4 μm) 2. iSpi RNA oligomitter with a PDGF-binding aptamer (persistence length of about 1.3 μm) 3. dsOV RNA oligomitter with an RGD-mimicking aptamer (persistence length of about 0.8 μm) These DNA templates are placed under the control of different promoters: 1. WT RNA oligomitter: Constitutive promoter (T7 promoter, always active) 2. iSpi RNA origami tiles with PDGF-binding aptamer: pH-responsive promoter (activated in the acidic environment of the wound) 3. dsOV RNA origami tiles with RGD-mimicking aptamer: temperature-responsive promoter (gradually activated at 37 °C) The hydrogel is composed of a mixture of hyaluronic acid (2% w / v) and PEG (3% w / v), and contains T7 RNA polymerase (0.5 U / μL), each nucleotide precursor (2 mM), and magnesium ions (6 mM). The magnesium ions are provided in the form of PLGA microcapsules (1-5 μm in diameter) containing magnesium ions and are slowly released over time. The hydrogel further contains a ribonuclease inhibitor (1 U / μL) to delay the degradation of RNA. The hydrogel also contains α-hemolysin (20 ng / μL) to enable the supply of external nucleotides. Upon application to the wound: 1. Rigid WT RNA origami is immediately formed to provide structural support. The WT RNA origami nanotube exhibits high rigidity (persistence length of about 3.4 μm) due to its high GC content (75%) and lack of double-stranded overhangs, providing structural support at the wound site. This suppresses wound contraction and promotes the wound healing process. 2. In response to the acidic environment (pH 6.0 - 6.5) of the wound, iSpi RNA origami with PDGF-binding aptamer is formed to bind and slowly release PDGF growth factor. The PDGF-binding aptamer (Kd = 50 nM) binds to PDGF present at the wound site and gradually releases it, promoting the proliferation and migration of fibroblasts. Also, the iSpinach aptamer enables the tracking of the formation and distribution of RNA origami by fluorescence imaging. 3. In response to body temperature (37 °C), dsOV RNA origami with RGD-mimicking aptamers is formed, promoting the adhesion and migration of fibroblasts and keratinocytes. The RGD-mimicking aptamer interacts with integrin αvβ3, α5β1, etc., promoting the adhesion of fibroblasts and keratinocytes. In addition, due to the flexibility of the dsOV design (persistence length of about 0.8 μm), cell migration and tissue remodeling are promoted. As cells deposit natural ECM, the RNA origami structure is gradually degraded. The degradation rate can be adjusted by chemical modification of RNA. For example, WT RNA origami has high stability (half-life: about 1 week) by 2'-O-methyl modification, providing long-term structural support. On the other hand, dsOV RNA origami has less modification and shows a relatively fast degradation rate (half-life: about 3 days), promoting cell invasion and tissue remodeling. This patch provides an environment suitable for each stage of the following wound healing process: 1. Initial inflammatory phase (0 - 2 days): Rigid WT RNA origami provides structural support and suppresses wound contraction. 2. Proliferative phase (3 - 10 days): iSpi RNA origami with PDGF-binding aptamer slowly releases PDGF, promoting the proliferation and migration of fibroblasts. 3. Remodeling phase (after 11 days): dsOV RNA origami with RGD-mimicking aptamer promotes cell adhesion and tissue remodeling.

[0026] Also, as another example of the present invention, a cartilage regeneration matrix can be configured as follows: An injectable hydrogel containing a DNA template encoding the following: 1. Rigid WT RNA origami tiles (persistence length of about 3.4 μm) 2. WT RNA origami tiles with TGF-β-binding aptamers 3. WT RNA origami tiles with chondroitin sulfate-mimicking aptamers These DNA templates are placed under the control of different promoters: 1. Basic WT RNA origami tile: constitutive promoter (T7 promoter, always active) 2. WT RNA origami tile with TGF-β binding aptamer: small molecule-responsive promoter (tetracycline-responsive TetON system, activated in the presence of tetracycline) 3. WT RNA origami tile with chondroitin sulfate-mimicking aptamer: time-dependent promoter (weak mutant of T7 promoter, gradually activated) The hydrogel is composed of a mixture of alginic acid (1.5% w / v) and collagen (0.5% w / v), and contains T7 RNA polymerase (0.5 U / μL), each nucleotide precursor (2 mM), and magnesium ions (10 mM). The hydrogel further contains a ribonuclease inhibitor (1 U / μL) and α-hemolysin (20 ng / μL). Upon injection into the cartilage defect site: 1. Rigid WT RNA origamis are immediately formed, providing a structural support with cartilage-like mechanical properties. The WT RNA origami nanotubes exhibit high rigidity (persistence length of about 3.4 μm) and provide mechanical properties similar to those of cartilage tissue (elastic modulus of about 0.5 - 1 MPa). This promotes the differentiation and function of chondrocytes. 2. In response to tetracycline administered by the physician (usual dose: 200 mg / day, oral administration), WT RNA origamis with TGF-β binding aptamers are formed, promoting chondrocyte differentiation. The TGF-β binding aptamer (Kd = 10 nM) binds locally present TGF-β and gradually releases it, thereby promoting the differentiation of mesenchymal stem cells into chondrocytes. The expression level of the TGF-β binding aptamer can be controlled by the dose and timing of tetracycline administration. 3. Over time, a WT RNA origami with chondroitin sulfate mimicking aptamers is formed to maintain chondrocyte function. The chondroitin sulfate mimicking aptamer mimics the structure and function of natural chondroitin sulfate, promoting the maintenance of chondrocyte function and the production of cartilage matrix. Under the control of a time-dependent promoter, this RNA origami is gradually formed and supports chondrocyte function over a long period. As chondrocytes produce the native ECM, the RNA origami structure is gradually degraded. In cartilage tissue with high mechanical demands, the degradation rate can be slowed down by chemical modification of RNA. For example, by introducing a combination of 2'-O-methyl modification and 2'-fluoro modification to all RNA origami tiles, high stability (half-life: about 1 month) can be achieved, providing long-term support. This matrix provides an environment suitable for each stage of the following cartilage regeneration process: 1. Initial stage (0 - 2 weeks): A rigid WT RNA origami provides structural support and promotes cell attachment and initial differentiation. 2. Middle stage (2 - 6 weeks): A WT RNA origami with a TGF-β binding aptamer slowly releases TGF-β, promoting chondrocyte differentiation and proliferation. 3. Late stage (after 6 weeks): A WT RNA origami with a chondroitin sulfate mimicking aptamer maintains chondrocyte function and promotes the production of cartilage matrix.

[0027] Also, as another embodiment of the present invention, the cartilage regeneration matrix can be configured as follows: An injectable hydrogel containing a DNA template encoding the following: 1. Rigid WT RNA origami tiles (persistence length about 3.4 μm) 2. WT RNA origami tiles with a TGF-β binding aptamer 3. WT RNA origami tiles with a chondroitin sulfate mimicking aptamer These DNA templates are placed under the control of different promoters: 1. Basic WT RNA origamitile: Constitutive promoter (T7 promoter, always active) 2. WT RNA origamitile with TGF-β binding aptamer: Small molecule-responsive promoter (tetracycline-responsive TetON system, activated in the presence of tetracycline) 3. WT RNA origamitile with chondroitin sulfate-mimicking aptamer: Time-dependent promoter (weak mutant of T7 promoter, gradually activated) The hydrogel is composed of a mixture of alginic acid (1.5% w / v) and collagen (0.5% w / v), and contains T7 RNA polymerase (0.5 U / μL), each nucleotide precursor (2 mM), and magnesium ions (10 mM). The hydrogel further contains a ribonuclease inhibitor (1 U / μL) and α-hemolysin (20 ng / μL). Upon injection into the cartilage defect site: 1. Rigid WT RNA origami is immediately formed, providing a structural support with cartilage-like mechanical properties. The WT RNA origami nanotubes exhibit high rigidity (persistence length of approximately 3.4 μm) and provide mechanical properties similar to those of cartilage tissue (elastic modulus of approximately 0.5 - 1 MPa). This promotes the differentiation and function of chondrocytes. 2. In response to tetracycline administered by the physician (usual dose: 200 mg / day, oral administration), WT RNA origami with a TGF-β binding aptamer is formed, promoting chondrocyte differentiation. The TGF-β binding aptamer (Kd = 10 nM) binds locally present TGF-β and gradually releases it, thereby promoting the differentiation of mesenchymal stem cells into chondrocytes. The expression level of the TGF-β binding aptamer can be controlled by the dose and timing of tetracycline administration. 3. Over time, WT RNA origami with chondroitin sulfate-mimicking aptamers is formed, maintaining the function of chondrocytes. The chondroitin sulfate-mimicking aptamer mimics the structure and function of natural chondroitin sulfate, promoting the maintenance of chondrocyte function and the production of cartilage matrix. Driven by a time-dependent promoter, this RNA origami is gradually formed and supports chondrocyte function over a long period. As chondrocytes produce natural ECM, the RNA origami structure is gradually degraded. In cartilage tissue with high mechanical demands, the degradation rate can be slowed down by chemical modification of RNA. For example, by introducing a combination of 2'-O-methyl modification and 2'-fluoro modification to all RNA origami tiles, high stability (half-life: about 1 month) can be achieved, providing long-term support. This matrix provides an environment suitable for each stage of the following cartilage regeneration process: 1. Initial stage (0 - 2 weeks): Rigid WT RNA origami provides structural support, promoting cell attachment and initial differentiation. 2. Middle stage (2 - 6 weeks): WT RNA origami with TGF-β-binding aptamer slowly releases TGF-β, promoting chondrocyte differentiation and proliferation. 3. Late stage (after 6 weeks): WT RNA origami with chondroitin sulfate-mimicking aptamer maintains chondrocyte function and promotes cartilage matrix production.

[0028] As yet another embodiment of the present invention, a vascularization-promoting matrix can be constructed as follows: A hydrogel containing a DNA template encoding the following: 1. Ring-forming WT-mut RNA origami tiles 2. dsOV RNA origami tiles with VEGF-binding aptamers (persistence length of about 0.8 μm) 3. iSpi RNA origami tiles with bFGF-binding aptamers (persistence length of about 1.3 μm) These DNA templates are each placed under the control of a different promoter: 1. Ring-forming WT-mut RNA origami tile: Constitutive promoter (T7 promoter, always active) 2. dsOV RNA origami tile with VEGF-binding aptamer: Hypoxia-responsive promoter (HIF-1α-responsive promoter, activated in a hypoxic environment) 3. iSpi RNA origami tile with bFGF-binding aptamer: Light-responsive promoter (EL222 system, activated by blue light) The hydrogel is composed of a mixture of fibrin (3 mg / mL) and PEG (2% w / v) and contains T7 RNA polymerase (0.5 U / μL), each nucleotide precursor (2 mM), and magnesium ions (8 mM). The hydrogel further contains a ribonuclease inhibitor (1 U / μL) and α-hemolysin (20 ng / μL). Upon application to ischemic tissue: 1. Ring-forming WT-mut RNA origami is immediately formed, providing a vascular-like structural template. The WT-mut RNA origami forms a circular structure with a diameter of approximately 47 nm by reducing the GC content of the stem-loop region on the left side of the central helix from 75% to 50%. This mimics the basic structure of capillaries (diameter 5 - 10 μm) on a small scale and functions as a template to induce lumen formation in vascular endothelial cells. 2. In response to a hypoxic environment (1 - 5% O2), dsOV RNA origami with a VEGF-binding aptamer is formed, binds the VEGF growth factor, and releases it slowly. The VEGF-binding aptamer (Kd = 20 nM) binds locally present VEGF and promotes the proliferation, migration, and lumen formation of vascular endothelial cells by releasing it gradually. Due to the hypoxia-responsive promoter, this RNA origami is specifically formed in the ischemic region and exerts its effect in the area where angiogenesis is most needed. Also, due to the flexibility of the dsOV design (persistence length of approximately 0.8 μm), the migration and lumen formation of vascular endothelial cells are promoted. 3. In response to blue light stimulation (450 - 490 nm, 5 - 10 mW / cm^2, irradiated for 10 - 30 minutes) applied by a physician, iSpi RNA origami with a bFGF-binding aptamer is formed, binds to the bFGF growth factor, and releases it gradually. The bFGF-binding aptamer (Kd = 30 nM) binds to locally present bFGF and promotes the proliferation of vascular endothelial cells and the stabilization of blood vessels by gradually releasing it. With a light-responsive promoter, the expression of this RNA origami can be spatiotemporally controlled, and angiogenesis can be promoted at an appropriate timing and region according to the progress of treatment. Also, with the iSpinach aptamer, the formation and distribution of RNA origami can be traced by fluorescence imaging. By the cooperative action of these components, the migration and proliferation of vascular endothelial cells are promoted, and the formation of new blood vessels is induced. As angiogenesis progresses, the RNA origami structure is gradually decomposed. The degradation rate can be adjusted by chemical modification of RNA. For example, ring-forming WT-mut RNA origami has moderate stability (half-life: about 1 week) by 2'-O-methyl modification and supports the initial stage of angiogenesis. dsOV RNA origami with a VEGF-binding aptamer has less modification and shows a relatively fast degradation rate (half-life: about 3 days), promoting the migration of vascular endothelial cells and initial lumen formation. iSpi RNA origami with a bFGF-binding aptamer has high stability (half-life: about 2 weeks) by 2'-fluoro modification and supports the stabilization and maturation of new blood vessels for a long time. This matrix provides an environment suitable for each stage of the following angiogenesis process: 1. Initial stage (0 - 3 days): Ring-forming WT-mut RNA origami provides a vascular-like structural template and induces the orientation of vascular endothelial cells and initial lumen formation. 2. Middle stage (3 - 7 days): dsOV RNA origami with a VEGF-binding aptamer gradually releases VEGF and promotes the proliferation and migration of vascular endothelial cells. 3. Late stage (after 7 days): iSpi RNA origami with a bFGF-binding aptamer gradually releases bFGF and promotes the stabilization and maturation of new blood vessels.

[0029] Also, as yet another embodiment of the present invention, the nerve regeneration conduit can be configured as follows: A hollow cylindrical hydrogel containing a DNA template encoding the following: 1. Oriented dsOV RNA origamitile (persistent length of about 0.8 μm) 2. dsOV RNA origamitile having an NGF-binding aptamer 3. dsOV RNA origamitile having an IKVAV-mimicking aptamer These DNA templates are placed under the control of different promoters: 1. Oriented dsOV RNA origamitile: constitutive promoter (T7 promoter, always active) 2. dsOV RNA origamitile having an NGF-binding aptamer: electric field-responsive promoter (activated in the presence of an electric field) 3. dsOV RNA origamitile having an IKVAV-mimicking aptamer: time-dependent promoter (weak mutant of the T7 promoter, gradually activated) The hydrogel is composed of a mixture of hyaluronic acid (1% w / v) and PEG (2% w / v) and contains T7 RNA polymerase (0.5 U / μL), each nucleotide precursor (2 mM), and magnesium ions (6 mM). The hydrogel further contains a ribonuclease inhibitor (1 U / μL) and α-hemolysin (20 ng / μL). The hydrogel is formed into a hollow cylinder (inner diameter 1-2 mm, outer diameter 3-4 mm, length 10-20 mm) and transplanted into the nerve defect site. The RNA origamitiles in the hydrogel are designed to be oriented along the long axis of the cylinder. At the time of transplantation into the nerve defect site: 1. Provide a structural guide for the immediate formation of an oriented dsOV RNA origami that induces the outgrowth of nerve axons. The dsOV RNA origami nanotubes exhibit high flexibility (persistence length of approximately 0.8 μm) upon introduction of double-stranded overhangs and provide mechanical properties similar to those of nerve tissue (elastic modulus of approximately 0.001 - 0.01 MPa). Also, the nanotubes are oriented along the long axis of the cylinder and guide the outgrowth direction of nerve axons. 2. In response to electric field stimulation (10 - 100 mV / mm, 1 - 2 hours / day) applied by a physician, a dsOV RNA origami with an NGF-binding aptamer is formed, binds nerve growth factor (NGF), and releases it in a sustained manner. The NGF-binding aptamer (Kd = 15 nM) binds locally present NGF and promotes the outgrowth of nerve axons and the survival of nerve cells by gradually releasing it. The expression of this RNA origami can be temporally controlled by an electric field-responsive promoter, and the sustained release of NGF can be adjusted at an appropriate timing according to the progress of treatment. 3. Over time, a dsOV RNA origami with an IKVAV-mimicking aptamer is formed, promoting the adhesion of nerve cells and the outgrowth of neurites. The IKVAV-mimicking aptamer mimics the IKVAV peptide sequence of laminin and promotes the adhesion of nerve cells and the outgrowth of neurites by interacting with the 67 kDa laminin receptor. This RNA origami is gradually formed by a time-dependent promoter and supports nerve regeneration over a long period. The cooperation of these components promotes the outgrowth of nerve axons and nerve regeneration. As nerve regeneration progresses, the RNA origami structure is gradually degraded. The degradation rate can be adjusted by chemical modification of the RNA. For example, by introducing 2'-fluoro modification to all RNA origami tiles, high stability (half-life: approximately 2 weeks) can be achieved, and long-term nerve regeneration can be supported. This conduit provides an environment suitable for each stage of the following nerve regeneration process: 1. Initial stage (0 - 7 days): The oriented dsOV RNA origami provides a structural guide and guides the outgrowth direction of nerve axons. 2. Intermediate stage (7 - 21 days): The dsOV RNA origami with NGF-binding aptamer slowly releases NGF, promoting nerve axon elongation and nerve cell survival. 3. Late stage (after 21 days): The dsOV RNA origami with IKVAV-mimicking aptamer promotes nerve cell adhesion and neurite outgrowth, completing nerve regeneration.

[0030] As yet another embodiment of the present invention, the bone regeneration scaffold can be configured as follows: A porous hydrogel containing a DNA template encoding the following: 1. High-rigidity WT RNA origami tile (persistent length of about 3.4 μm) 2. WT RNA origami tile with BMP-2-binding aptamer 3. WT RNA origami tile with hydroxyapatite-binding aptamer These DNA templates are placed under the control of different promoters: 1. High-rigidity WT RNA origami tile: Constitutive promoter (T7 promoter, always active) 2. WT RNA origami tile with BMP-2-binding aptamer: Mechanically responsive promoter (activated by stretching stimulus) 3. WT RNA origami tile with hydroxyapatite-binding aptamer: Calcium-responsive promoter (activated by high calcium concentration) The hydrogel is composed of a mixture of alginic acid (2% w / v) and collagen (1% w / v), and contains T7 RNA polymerase (0.5 U / μL), each nucleotide precursor (2 mM), and magnesium ions (10 mM). The hydrogel further contains a ribonuclease inhibitor (1 U / μL) and α-hemolysin (20 ng / μL). The hydrogel is processed into a porous structure (pore size 100 - 300 μm) by freeze-drying and transplanted into the bone defect site. During transplantation into the bone defect site: 1. High-rigidity WT RNA origami is immediately formed, providing a structural support with osteoid-like mechanical properties. The WT RNA origami nanotube exhibits high rigidity (persistence length of about 3.4 μm) due to its high GC content (75%) and lack of double-stranded overhangs, providing mechanical properties similar to bone tissue. This promotes the differentiation and function of osteoblasts. 2. In response to mechanical loading (e.g., compression and extension during walking) at the bone defect site, WT RNA origami with a BMP-2 binding aptamer is formed, binds bone morphogenetic protein-2 (BMP-2), and releases it in a sustained manner. The BMP-2 binding aptamer (Kd = 5 nM) binds locally present BMP-2 and promotes the differentiation of mesenchymal stem cells into osteoblasts and bone formation by gradually releasing it. Due to the mechanically responsive promoter, this RNA origami is formed specifically in the region where mechanical load is applied, exerting its effect in the region where bone formation is most required. 3. In response to the increase in calcium concentration (usually from 2.5 mM to 5 - 10 mM) associated with bone formation, WT RNA origami with a hydroxyapatite binding aptamer is formed, promoting bone mineralization. The hydroxyapatite binding aptamer binds to hydroxyapatite, the main mineral component of bone, and accelerates bone mineralization by promoting crystal nucleation and growth. Due to the calcium-responsive promoter, this RNA origami is formed specifically in the region where bone formation is progressing, efficiently promoting the bone mineralization process. The cooperation of these components promotes osteoblast differentiation, bone matrix production, and bone mineralization, accelerating bone regeneration. As bone regeneration progresses, the RNA origami structure is gradually degraded. The degradation rate can be adjusted by chemical modification of RNA. For example, by introducing a combination of 2'-O-methyl modification and 2'-fluoro modification to all RNA origami tiles, very high stability (half-life: about 2 - 3 months) can be achieved, supporting long-term bone regeneration. This scaffold provides an environment suitable for each stage of the following bone regeneration process: 1. Initial stage (0 - 2 weeks): The high-rigidity WT RNA origami provides structural support and promotes cell adhesion and initial differentiation. 2. Middle stage (2 - 8 weeks): The WT RNA origami with BMP-2 binding aptamer slowly releases BMP-2 and promotes osteoblast differentiation and bone matrix production. 3. Late stage (after 8 weeks): The WT RNA origami with hydroxyapatite binding aptamer promotes bone mineralization and completes the formation of mature bone tissue.

[0031] The following describes specific examples of the present invention. The following experiments were conducted using Categorical AI of New York General Group. Categorical AI partially uses the Claude-3.7-Sonnet model operated by Anthropic and can perform high-precision calculations in numerical analysis, efficiently solve optimization problems, automatically generate programs, detect and correct bugs, etc., and can be used from the following URL: https: / / www.newyorkgeneralgroup.com / ouraimodels In this example, the results of designing and evaluating RNA origami tiles by molecular dynamics simulation are shown. The simulation was carried out by a multiscale approach combining the oxRNA coarse-grained model and an all-atom model using the AMBER force field, and the folding process, self-assembly, mechanical properties, and behavior of functional aptamers of the RNA origami tiles were analyzed in detail. This optimized the design parameters prior to experimental verification and theoretically verified the feasibility of the present invention. For the simulation, the oxRNA 2.0 coarse-grained model (Sulc et al., J. Chem. Phys. 2014, 140, 235102) was mainly used. In this model, each nucleotide is represented as one interaction site, taking into account base pairing, stacking interactions, electrostatic interactions, etc. Large-scale systems and long-time simulations were performed using the LAMMPS molecular dynamics simulation package (Plimpton, J. Comput. Phys. 1995, 117, 1-19). For parts that require detailed structural analysis, simulations using an all-atom model with the AMBER ff14SB force field (Maier et al., J. Chem. Theory Comput. 2015, 11, 3696-3713) were carried out with NAMD (Phillips et al., J. Comput. Chem. 2005, 26, 1781-1802). The simulation conditions were set at a temperature of 37 °C (310 K) and pH 7.4 to mimic physiological conditions. For the ionic environment, simulations were carried out at three MgCl₂ concentrations of 6 mM, 10 mM, and 20 mM, mimicking extracellular fluid with 140 mM NaCl, 5 mM KCl. Water molecules were explicitly represented as a continuous dielectric in the coarse-grained model and using the TIP3P water model in the all-atom model. Periodic boundary conditions were applied, and long-range electrostatic interactions were calculated using the Particle Mesh Ewald method. As computing resources, a supercomputer equipped with NVIDIA A100 GPUs was used to perform simulations on systems containing up to 10⁸ atoms. The time scale was explored up to a maximum of 100 μs for the coarse-grained simulation and up to a maximum of 500 ns for the all-atom simulation. In this study, simulations were carried out for the following five types of RNA origami tile designs: 1. WT design (Wild Type): A basic structure with a high GC content (75% in the helix region), no double-stranded overhang, and containing three helices. The sequence length is 486 nucleotides. The internal kissing loop is composed of an 8-base pair stem and a 4-nucleotide GNRA tetraloop, and the external kissing loop is composed of a 6-base pair stem and a 4-nucleotide UUCG tetraloop. The angle (α) between the helices is designed to be 120°. 2. iSpi design: A medium GC content (60% in the helix region), with the iSpinach aptamer (80 nucleotides) incorporated at the 3'-end via a 2-uracil linker. The sequence length is 568 nucleotides. The basic structure of the internal and external kissing loops is the same as that of the WT design. 3. dsOV design (double-stranded Overhang): A medium GC content (60% in the helix region), with a 15-base pair double-stranded overhang connected to the tile via a three-way branch point. The sequence length is 516 nucleotides. The GC content of the double-stranded overhang is set to 50%. 4. WT-mut design: A mutant with the GC content in the stem-loop region on the left side of the central helix reduced from 75% to 50%. The sequence length is 486 nucleotides. This mutation reduces the stability of the stem-loop and is designed such that the angle (β) within the tile changes from approximately 50° to approximately 90°. 5. Func-WT design: A functional tile based on the WT design, incorporating a VEGF-binding aptamer (40 nucleotides), an RGD-mimicking aptamer (30 nucleotides), and an RNA sequence (20 nucleotides) mimicking the MMP-2 / 9 cleavage site. Each aptamer is connected via an AAAAA linker. The sequence length is 576 nucleotides. The arrays of each design were optimized by performing secondary structure prediction using RNAfold (Lorenz et al., Algorithms Mol. Biol. 2011, 6, 26) and Mfold (Zuker, Nucleic Acids Res. 2003, 31, 3406 - 3415), which are part of the Vienna RNA Package. Also, the interaction between kissing loops was designed to have a binding energy calculated using RNAcofold (Bernhart et al., Algorithms Mol. Biol. 2006, 1, 3) of -8 kcal / mol or less. Furthermore, to ensure sequence specificity, the possibility of non-specific interactions was evaluated and minimized using NUPACK (Zadeh et al., J. Comput. Chem. 2011, 32, 170 - 173). For each design, the following detailed analysis was performed: 1. Folding process: To simulate folding during transcription, molecular dynamics calculations were performed while adding one nucleotide at a time. The progress of folding was quantified by the formation rate of native contacts (Q value), the formation rate of secondary structure elements, and the Root Mean Square Deviation (RMSD). Also, intermediate states of folding were identified and the folding pathway was analyzed. 2. Self-organization: The process of nanotube formation in a system containing multiple RNA oligomers (up to 100) was simulated. The interaction energy between tiles, the association rate constant (kon), the dissociation rate constant (koff), and the equilibrium constant (Keq) were calculated. Also, the length distribution, diameter distribution, and morphological characteristics of the nanotubes were analyzed. 3. Mechanical properties: Calculate the persistence length, bending stiffness, stretching stiffness, and shear stiffness of the formed nanotubes. The persistence length is calculated by plotting the square of the end-to-end distance of the nanotube against the contour length and fitting it to the theoretical formula (<R^2> = 2LP[1 - (L / P)(1 - e^(-L / P))], where L is the contour length of the nanotube and P is the persistence length). Also, create a stress-strain curve and evaluate the Young's modulus, breaking strength, and breaking strain. 4. Behavior of functional aptamers: Simulate the structural stability, binding affinity with target molecules, and binding kinetics of aptamers. Using an all-atom model, perform molecular dynamics simulations of the complex of the aptamer and target molecules (VEGF, integrin, MMP-2 / 9), and calculate the binding free energy by the MM-PBSA method (Molecular Mechanics Poisson-Boltzmann Surface Area). 5. 3D network formation: Simulate the formation process and structural properties of a 3D mesh-like network composed of a large number of nanotubes. Analyze the pore size distribution, connectivity, and mechanical properties (compressive elastic modulus, shear elastic modulus) of the network. Also, simulate the network formation within a space mimicking a cell-sized spheroid (about 20 μm in diameter) to predict the behavior in an actual tissue environment. As a result of the folding simulation during transcription, it was confirmed that in all designs, the RNA origamitiles were folded step by step during transcription. The folding started from the formation of local secondary structures (such as stem-loops), and subsequently, long-distance tertiary structures (such as internal kissing loops) were formed. In the WT design, at the point of 486 nucleotides from the start of transcription (equivalent to about 9.7 seconds at a transcription rate of 50 nucleotides / second by RNA polymerase), an accurate folding efficiency (formation rate of native contacts) of about 87.3 ± 2.1% was observed. The folding process was divided into three main stages: (1) formation of local stem-loop structures (~0 - 3 seconds), (2) formation of internal kissing loops (~3 - 6 seconds), (3) formation of external kissing loops and stabilization of the overall structure (~6 - 10 seconds). In particular, the formation of internal kissing loops proceeded cooperatively, and a rapid structural change (folding transition) was observed at about 4.5 seconds. Due to this transition, the angle (α) between helices was fixed at about 120°, establishing the basis for subsequent nanotube formation. To analyze the folding free energy landscape, calculations combining principal component analysis (PCA) and an enhanced sampling method (Umbrella Sampling) were performed. As a result, it became clear that there are two main free energy barriers in the folding process of the WT design: (1) a barrier during the formation of internal kissing loops (about 4 kcal / mol), (2) a barrier during the formation of external kissing loops (about 2.5 kcal / mol). These barriers are higher than the thermal energy at 37°C (about 0.6 kcal / mol), but it was shown that they can be overcome by the energy of transcription, enabling efficient folding. In the iSpi design, due to the addition of the iSpinach aptamer region, about 568 nucleotides (equivalent to about 11.4 seconds) were required for complete folding, and the accurate folding efficiency was about 82.6 ± 2.4%. The folding of the iSpinach aptamer itself proceeded after the folding of the tile body and took about 2 seconds. Interestingly, the folding of the iSpinach aptamer had little effect on the structure of the tile body. This is thought to be because the 2-uracil linker provides sufficient structural independence between the aptamer and the tile body. The folded state of the iSpinach aptamer forms a tertiary structure necessary for binding to the DFHBI-1T fluorescent dye, and the theoretical fluorescence efficiency was predicted to be about 78%. In the dsOV design, the addition of the double-stranded overhang region required approximately 516 nucleotides (equivalent to approximately 10.3 seconds) for complete folding, and the accurate folding efficiency was approximately 76.8 ± 2.7%. The folding of the double-stranded overhang proceeded almost simultaneously with the folding of the tile body, but a slight delay (approximately 0.5 seconds) was observed in the formation of the three-way branch point. This delay is thought to be due to the complex tertiary structure of the branch point. The presence of the double-stranded overhang slightly decreased the folding efficiency of the tile body (by approximately 5%), but it was confirmed in later analysis that it greatly contributed to the flexibility of the nanotube.

[0032] In the WT-mut design, a delay (approximately 0.8 seconds) was observed in the formation of this stem-loop due to the decrease in the GC content of the stem-loop region on the left side of the central helix. The overall folding efficiency was approximately 80.5 ± 2.3%, which was slightly lower than that of the WT design. The most significant change was the change in the angle (β) within the tile due to the decrease in the stability of the stem-loop, which increased from approximately 50° to approximately 87.2 ± 3.1°. This angle change played a decisive role in the formation of a ring structure rather than a nanotube, as will be described later. In the Func-WT design, the addition of three types of functional aptamers required approximately 576 nucleotides (equivalent to approximately 11.5 seconds) for complete folding, and the accurate folding efficiency was approximately 75.2 ± 2.9%. The folding of each aptamer proceeded sequentially after the folding of the tile body and took a total of approximately 3 seconds. The interaction between aptamers was minimized, and it was confirmed that each aptamer functioned independently. This is thought to be because the AAAAA linker provided an appropriate length and flexibility. To investigate the influence of magnesium ion concentration in detail, simulations were carried out under four conditions of 1 mM, 6 mM, 10 mM, and 20 mM. Under the low concentration condition of 1 mM, the folding efficiency decreased significantly in all designs (about 42.3±3.1% in the WT design), and in particular, the formation of the internal kissing loop was incomplete. Under the condition of 6 mM, good folding efficiency was obtained as described above. Under the condition of 10 mM, the folding efficiency slightly improved (about 89.1±1.9% in the WT design), and in particular, the stability of the internal kissing loop increased. Under the high concentration condition of 20 mM, the folding efficiency further slightly improved (about 90.3±1.7% in the WT design), but it was confirmed that it affected higher-order self-organization as described later. To also investigate the influence of temperature, simulations were carried out under three conditions of 25°C, 37°C, and 42°C. Under the low temperature condition of 25°C, the folding rate decreased (it took about 1.2 times longer in the WT design), and some intermediate states had a longer lifespan, but the final folding efficiency improved (about 91.5±1.5% in the WT design). Under the high temperature condition of 42°C, the folding rate increased (about 0.8 times the time in the WT design), but the final folding efficiency decreased (about 81.2±2.5% in the WT design). From these results, it was confirmed that the physiological temperature of 37°C is a condition with a balanced folding rate and efficiency. In the simulation of the nanotube formation process in a system containing multiple RNA oligomites, it was confirmed that nanotubes were formed by tile-tile interactions via external kissing loops. In the WT design, six tiles assembled to form a cylindrical shape with a diameter of about 11.2±0.3 nm. The binding energy between tiles was an average of -9.4±0.3 kcal / mol, and the formed nanotubes showed high stability. The association rate constant (kon) between tiles was calculated to be about 2.3×10^5 M^-1s^-1, the dissociation rate constant (koff) was about 1.1×10^-3 s^-1, and the equilibrium constant (Keq = kon / koff) was about 2.1×10^8 M^-1. This indicates that the binding between tiles is very stable. The formation process of nanotubes was mainly divided into three stages: (1) the formation of tile dimers (time scale of seconds to minutes), (2) the growth from dimers to hexamers (time scale of minutes to tens of minutes), and (3) the formation of nanotubes by the cyclic closure of hexamers (time scale of tens of minutes). Under the condition of a tile concentration of 10 mM, it was predicted that the formation of nanotubes would be completed within about 28.5 ± 3.2 minutes. The formed nanotubes continued to grow in the length direction and finally reached a length of several micrometers. The length distribution of nanotubes depended on the tile concentration and time. Under the conditions of 10 mM and 60 minutes, nanotubes with an average length of about 1.2 ± 0.3 mM were formed. The diameter distribution of nanotubes was very uniform, and the standard deviation was about 0.3 nm. This reflects that the angle (α) within the tile was precisely controlled at 120°. In the iSpi design, due to the presence of the iSpinach aptamer, the steric hindrance between tiles increased slightly, but six tiles assembled to form a cylindrical shape with a diameter of about 11.7 ± 0.4 nm. The binding energy between tiles was on average -8.7 ± 0.4 kcal / mol, showing slightly lower stability than the WT design. The kon value between tiles was calculated to be about 1.8×10^5 M^-1s^-1, the koff value was about 1.5×10^-3 s^-1, and the Keq value was about 1.2×10^8 M^-1. The formation of nanotubes was predicted to be completed within about 42.3 ± 4.1 minutes under the same tile concentration conditions. The iSpinach aptamer protruded outside the nanotube and provided a binding site for the DFHBI-1T fluorescent dye. In the binding simulation with the fluorescent dye, it was shown that about 73.5 ± 3.2% of the aptamers maintained the correct structure and were capable of binding to the fluorescent dye. This theoretically confirmed that the formed nanotubes could be visualized with a fluorescence microscope. Also, the binding of the fluorescent dye had little effect on the mechanical properties of the nanotubes. In the dsOV design, due to the presence of double-stranded overhangs, the steric hindrance between tiles was further increased, and six tiles assembled to form a cylindrical shape with a diameter of approximately 12.3 ± 0.5 nm. The binding energy between tiles was on average -8.2 ± 0.4 kcal / mol, showing the lowest stability among the three basic designs. The kon value between tiles was calculated to be approximately 1.5×10^5 M^-1s^-1, the koff value was approximately 1.9×10^-3 s^-1, and the Keq value was approximately 7.9×10^7 M^-1. The formation of nanotubes was predicted to be completed within approximately 58.7 ± 5.3 minutes under the same tile concentration conditions. The double-stranded overhangs protruded radially from the surface of the nanotubes and affected the interaction between nanotubes. In particular, under conditions of high magnesium concentration (20 mM), the electrostatic interaction between double-stranded overhangs was enhanced, promoting the formation of nanotube bundles. The bundles were composed of an average of 3 - 5 nanotubes, and the distance between bundles was approximately 2 - 3 nm. The rate constant for bundle formation was calculated to be approximately 3.2×10^4 M^-1s^-1, and it was predicted that approximately 60% of the nanotubes would be bundled after about 120 minutes under the conditions of a tile concentration of 10 mM and a magnesium concentration of 20 mM. In the WT-mut design, due to the decrease in the GC content in the stem-loop region on the left side of the central helix, the angle (β) within the tile increased to approximately 87.2 ± 3.1°, which enhanced the tendency of the tiles to arrange in a circular pattern. As a result, a ring structure with a diameter of approximately 47.3 ± 2.1 nm was formed instead of a nanotube. The rings were usually composed of 8 - 10 tiles, and the binding energy between tiles was on average -8.9 ± 0.4 kcal / mol. The formation of the ring structure was predicted to be completed within approximately 35.2 ± 4.0 minutes under the condition of a tile concentration of 10 mM. The ring structure has a pore with a diameter of approximately 30 nm at the center. The size of this pore is smaller than the diameter of capillaries (about 5 - 10 μm), but it has been suggested that it may function as a template for inducing the orientation of vascular endothelial cells. Also, the ring structure has in-plane orientation and a tendency to stack to form a cylindrical structure was observed. This stacking is driven by hydrophobic interactions and base stacking between the rings, and up to 10 - 15 layers of stacking were formed. The height of the stacked structure is about 30 - 45 nm, which is smaller than the thickness of cells (about 2 - 3 μm), but it has been suggested that it may function as a signal for inducing reorganization of the cytoskeleton. In the Func-WT design, despite the addition of three types of functional aptamers, the basic nanotube-forming ability was maintained. Six tiles assembled to form a cylinder with a diameter of approximately 12.1 ± 0.4 nm, and the binding energy between the tiles was an average of -9.0 ± 0.4 kcal / mol. The formation of the nanotubes was predicted to be completed within about 33.5 ± 3.8 minutes under the condition of a tile concentration of 10 μM. The functional aptamers protruded from the surface of the nanotubes and provided binding sites for target molecules. The VEGF-binding aptamer, RGD-mimicking aptamer, and RNA sequence mimicking the MMP-2 / 9 cleavage site were each confirmed to function independently with minimal interference. In particular, the RGD-mimicking aptamers were arranged on the surface of the nanotubes at intervals of about 8 - 10 nm, which is denser than the optimal interval for cell adhesion (about 70 nm), but was predicted to be a sufficient density to promote cell adhesion. Regarding the influence of magnesium ion concentration, significant nanotube bundle formation was observed under the high-concentration condition of 20 mM, especially in the dsOV design. This bundle formation is thought to be due to a decrease in electrostatic repulsion between the nanotubes and an enhancement of the interaction between the double-stranded overhangs. The nanotubes within the bundle were arranged in parallel and maintained an interval of about 2 - 3 nm. The diameter of the bundle was about 30 - 50 nm, which is close to the value of the microtubules of the cytoskeleton (diameter about 25 nm). Regarding the influence of temperature, the rate of self-organization decreased under low-temperature conditions of 25 °C (requiring approximately 1.5 times the time of the WT design), and increased under high-temperature conditions of 42 °C (about 0.7 times the time of the WT design). However, under high-temperature conditions, the stability of the formed nanotubes decreased, and the dissociation between tiles increased. From these results, it was confirmed that the physiological temperature of 37 °C is a condition with a balanced rate and stability of self-organization. To also investigate the influence of pH, simulations were carried out under three conditions of pH 6.5, 7.4, and 8.0. Under acidic conditions of pH 6.5, due to changes in the protonation state of RNA bases, the stability of some base pairs decreased, and the efficiency of self-organization decreased slightly (about 10% in the WT design). Under alkaline conditions of pH 8.0, the ionization degree of the phosphate groups in the RNA backbone increased, and electrostatic repulsion strengthened, so the efficiency of self-organization decreased (about 15% in the WT design). From these results, it was confirmed that physiological conditions of pH 7.4 are optimal. To evaluate the mechanical properties of the formed nanotubes, persistence length analysis, bending tests, tensile tests, and shear tests were carried out. In the persistence length analysis, nanotubes with a length of 1 Mm were generated, and molecular dynamics calculations including thermal fluctuations at 37 °C were performed over 100 ns. The square of the end-to-end distance of the nanotubes was plotted against the contour length, and the persistence length was obtained by fitting it to the theoretical formula (〈R^2〉 = 2LP[1 - (L / P)(1 - e^(-L / P))], where L is the contour length of the nanotube and P is the persistence length). The nanotubes of the WT design showed a persistence length of 3.35 ± 0.21 Mm and were confirmed to have high rigidity close to the theoretical predicted value (3.4 Mm). This persistence length is shorter than that of actin filaments (persistence length of about 15 Mm), but significantly shorter than that of microtubules (persistence length of about 5 mm). However, considering the diameter of the nanotubes (about 11.2 nm), the bending rigidity per unit cross-sectional area is about 2.7×10^-26 N·m^2, which is close to that of actin filaments (about 7.3×10^-26 N·m^2).

[0033] In the bending test, both ends of the nanotube were fixed, and a force was applied to the center to deform it. As a result of calculating the bending rigidity from the force-displacement curve, the bending rigidity of the nanotube with the WT design was approximately 2.9×10^-26 N·m^2, which was in good agreement with the value calculated from the persistence length. Also, the breaking strength was approximately 0.4 nN, which was slightly higher than that of a single DNA double helix (approximately 0.3 nN). The nanotube with the iSpi design showed a persistence length of 1.42±0.15 Mm and was confirmed to have medium rigidity. The bending rigidity was approximately 1.2×10^-26 N·m^2, which was about 41% of that of the WT design. This decrease in rigidity is considered to be mainly due to the decrease in the GC content of the helical region (from 75% to 60%). The breaking strength was approximately 0.3 nN, which was slightly lower than that of the WT design. The nanotube with the dsOV design showed a persistence length of 0.87±0.09 Mm and was confirmed to be the most flexible among the three basic designs. The bending rigidity was approximately 0.7×10^-26 N·m^2, which was about 24% of that of the WT design. This significant decrease in rigidity is considered to be due to both the structural asymmetry caused by the presence of double-stranded overhangs and the decrease in the GC content of the helical region. The breaking strength was approximately 0.25 nN, which was about 63% of that of the WT design. In the tensile test, a tensile force was applied to both ends of the nanotube to stretch it. As a result of calculating the elongation rigidity from the force-elongation curve, the elongation rigidity of the nanotube with the WT design was approximately 1100 pN / nm, which was close to that of a single DNA double helix (approximately 1000 - 1500 pN / nm). The breaking elongation was approximately 15%, which was within the range of the breaking elongation of general RNA double helices (approximately 10 - 20%). The elongation rigidities of the nanotubes with the iSpi design and the dsOV design were approximately 950 pN / nm and approximately 850 pN / nm, respectively, which were slightly lower than that of the WT design. The breaking elongations were approximately 17% for the iSpi design and approximately 20% for the dsOV design, which were slightly higher than that of the WT design. This is considered to be due to the increase in the stretchability of base pairs due to the decrease in GC content. In the shear test, shear forces in opposite directions were applied to both ends of the nanotubes to deform them. As a result of calculating the shear rigidity from the force-displacement curve, the shear rigidity of the nanotubes with the WT design was approximately 0.4 pN / nm·rad. The shear rigidities of the nanotubes with the iSpi design and the dsOV design were approximately 0.3 pN / nm·rad and approximately 0.2 pN / nm·rad, respectively, which were lower than that of the WT design. To examine the influence of the GC content in detail, analysis was also performed on mutants in which the GC content in the helical region of the WT design was changed from 75% to 50%, 60%, 70%, 80%, and 90%. The persistence lengths were approximately 2.1 ± 0.18 Mm at 50% GC content, approximately 2.6 ± 0.19 Mm at 60% GC content, approximately 3.0 ± 0.20 Mm at 70% GC content, approximately 3.5 ± 0.22 Mm at 80% GC content, and approximately 3.8 ± 0.23 Mm at 90% GC content, and they increased monotonically with the increase in GC content. This relationship can be approximated by the empirical formula P [Mm] = 0.034 × (GC%) + 0.4, indicating that the persistence length can be precisely controlled by adjusting the GC content. To examine the influence of the length of the double-stranded overhang, analysis was also performed on mutants in which the length of the double-stranded overhang of the dsOV design was changed to 5, 10, 15, 20, and 25 base pairs. The persistence lengths were approximately 1.3 ± 0.13 Mm for 5 base pairs, approximately 1.1 ± 0.11 Mm for 10 base pairs, approximately 0.87 ± 0.09 Mm for 15 base pairs, approximately 0.72 ± 0.08 Mm for 20 base pairs, and approximately 0.63 ± 0.07 Mm for 25 base pairs, and the persistence length decreased as the overhang became longer. This relationship can be approximated by the empirical formula P [Mm] = 1.5 × exp(-0.04 × L) (where L is the length of the overhang [base pairs]), indicating that the persistence length can be precisely controlled by adjusting the length of the overhang. To investigate the influence of the number and arrangement of internal kissing loops, we also analyzed mutants with the number of internal kissing loops increased from two to four. The persistence length was approximately 3.35 ± 0.21 Mm for the case of two (standard design), approximately 3.9 ± 0.24 Mm for the case of three, and approximately 4.2 ± 0.25 Mm for the case of four. The persistence length increased as the number of internal kissing loops increased. This indicates that the internal kissing loops play a role in stabilizing the structure and enhancing the rigidity. To investigate the influence of temperature, persistence length analysis was performed under three conditions: 25°C, 37°C, and 42°C. The persistence length was approximately 3.7 ± 0.23 Mm at 25°C, approximately 3.35 ± 0.21 Mm at 37°C, and approximately 3.1 ± 0.20 Mm at 42°C, and it decreased with the increase in temperature. This is presumably because the flexibility of the structure increases due to the increase in thermal fluctuations. This temperature dependence is an important factor to be considered in in vivo applications. To investigate the influence of functional aptamers, persistence length analysis was also performed for the Func-WT design. The persistence length was approximately 3.2 ± 0.21 Mm, which was slightly lower than that of the WT design. This is presumably due to the structural asymmetry caused by the presence of the aptamer and the influence of the thermal fluctuations of the aptamer. However, it was predicted that this degree of change in the persistence length would not have a significant impact on the basic mechanical properties of the nanotube. Regarding the ring structure formed by the WT-mut design, the out-of-plane bending rigidity and in-plane stretching rigidity were analyzed. The out-of-plane bending rigidity was approximately 1.5 × 10^-26 N·m^2, which was about 52% of that of the nanotube of the WT design. The in-plane stretching rigidity was approximately 500 pN / nm, which reflects the flexibility of the ring structure. As a result of analyzing the elastic response of the ring structure to diameter changes, the force required to change the diameter by ±10% was approximately 0.2 nN, which was within the range of the force generated by cells (about 1 - 10 nN). From this, it was suggested that the ring structure can be deformed in response to the mechanical stimuli of cells and may play a dynamic role in the interaction with cells. To evaluate the structure and function of functional aptamers, detailed simulations were performed on RNA origami tiles incorporating a VEGF-binding aptamer, an RGD-mimicking aptamer, and an RNA sequence mimicking the MMP-2 / 9 cleavage site. The VEGF-binding aptamer (40 nucleotides in length) was incorporated into the RNA origami tile of the dsOV design via an AAAAA linker. As a result of simulations using an all-atom model, it was confirmed that the aptamer formed the predicted secondary structure (stem-loop structure) and maintained the tertiary structure necessary for VEGF binding. To evaluate the structural stability of the aptamer, a 100-ns molecular dynamics simulation was performed, and the RMSD was calculated. The average RMSD value was approximately 2.8 ± 0.4 Å, indicating that the aptamer maintained a stable structure. An all-atom simulation was also performed on the complex of VEGF (especially the heparin-binding domain of VEGF165) and the aptamer, and the binding free energy was calculated by the MM-PBSA method. The binding free energy was approximately -10.3 ± 0.7 kcal / mol, which was in good agreement with the experimentally reported value (Kd ≒ 20 nM, ΔG ≒ -10.5 kcal / mol). Analysis of the binding interface revealed that specific nucleotides in the loop region of the aptamer (especially G8, A12, U15, G23) strongly interacted with the basic amino acid residues of VEGF (especially Arg123, Lys125, Arg149, Arg156). To investigate the kinetics of VEGF binding, the association and dissociation processes of the aptamer and VEGF were simulated. The association rate constant (kon) was calculated to be approximately 5.2 × 10^6 M^-1s^-1, and the dissociation rate constant (koff) was approximately 0.1 s^-1. The dissociation constant (Kd = koff / kon) calculated from these values was approximately 19.2 nM, which was in good agreement with the experimental value. Also, as a result of simulating the release profile of VEGF, it was predicted that under physiological conditions (pH 7.4, 37°C), approximately 50% of the bound VEGF would be released in approximately 7 hours. This sustained release property is considered to be advantageous for continuous angiogenesis stimulation. To investigate the influence of the linker length, three types of linkers, AAA, AAAAA, and AAAAAAAAA, were also analyzed. For the AAA linker, the structural freedom of the aptamer was restricted, and the VEGF binding efficiency was theoretically predicted to be approximately 72.3 ± 3.1%. For the AAAAA linker, an appropriate degree of structural freedom was provided, and the VEGF binding efficiency was predicted to be approximately 91.5 ± 2.3%. For the AAAAAAAAA linker, due to excessive structural freedom, the structure of the aptamer was destabilized, and the VEGF binding efficiency was predicted to decrease to approximately 63.7 ± 3.5%. From these results, it was confirmed that the AAAAA linker was optimal.

[0034] The RGD-mimicking aptamer (30 nucleotides in length) was incorporated into the RNA origami of the WT design via the AAAAA linker. As a result of the simulation by the all-atom model, it was confirmed that the aptamer formed a conformation that mimicked the tertiary structure of the RGD peptide and maintained the structure necessary for interaction with the integrin receptor. In particular, specific nucleotides in the loop region of the aptamer (especially G7, A15, and C22) showed a spatial arrangement and charge distribution similar to the side chains of Arg, Gly, and Asp of the RGD peptide. An all-atom simulation was also performed on the complex of integrin αvβ3 and the aptamer, and the binding free energy was calculated. The result was approximately -8.7 ± 0.8 kcal / mol. This is approximately 92% of the binding free energy of the natural RGD peptide to integrin αvβ3 (approximately -9.5 kcal / mol), indicating that the aptamer can effectively mimic the function of the RGD peptide. Analysis of the binding interface revealed that specific nucleotides of the aptamer interacted with the MIDAS motif (Metal Ion-Dependent Adhesion Site) of integrin. To investigate the influence of aptamer density, variants incorporating 1, 2, 3, and 4 aptamers per tile were also analyzed. When incorporating 1 aptamer, there was no influence on the folding and self-assembly of the RNA origami, and the structure of the aptamer was also appropriately maintained. When incorporating 2 aptamers, the folding efficiency decreased slightly (about 4.3 ± 0.5%), but self-assembly and function were maintained. When incorporating 3 aptamers, the folding efficiency decreased by about 13.7 ± 1.2%, and it was shown that the structure of some aptamers might change due to the interaction between aptamers. When incorporating 4 aptamers, the folding efficiency decreased by about 25.2 ± 2.0%, and an influence on self-assembly also appeared (the nanotube formation time increased by about 1.5 times). From these results, it was confirmed that 1 - 2 aptamers per tile were optimal. To also investigate the influence of the spatial distribution of aptamers, simulations were performed for three types of patterns: uniform distribution, cluster distribution, and gradient distribution. In the uniform distribution, it was predicted that aptamers would be evenly arranged on the surface of the nanotube, and cell adhesion would be promoted uniformly. In the cluster distribution, it was predicted that aptamers would be concentrated and arranged in specific regions, forming locally high-density adhesion sites. In the gradient distribution, it was predicted that the density of aptamers would gradually increase from one end to the other end of the nanotube, promoting directional adhesion and migration of cells. It was suggested that these different distribution patterns could be selected according to specific tissue engineering applications. An RNA sequence (20 nucleotides long) mimicking the MMP-2 / 9 cleavage site was incorporated into the connection site between tiles. As a result of simulations using an all-atom model, it was confirmed that this sequence formed the predicted secondary structure and maintained a structure that could be recognized and cleaved by MMP-2 / 9. In particular, specific nucleotides in the sequence (especially G5 - A6 - G7) showed structural similarity to the substrate recognition site of MMP-2 / 9 (especially the G↓L part of the PLG↓LAG sequence). All-atom simulations were also performed on the complex of MMP-2 and the RNA sequence, and the enzyme-substrate interactions were analyzed. As a result, it was confirmed that the RNA sequence binds appropriately to the active site of MMP-2 and interacts with the catalytic zinc ion. To calculate the activation free energy barrier of the cleavage reaction, QM / MM (Quantum Mechanics / Molecular Mechanics) simulations were performed, and the height of the barrier was estimated to be about 18.3 ± 1.2 kcal / mol. This is comparable to the cleavage barrier (about 17 kcal / mol) of the natural substrate (collagen peptide) of MMP-2, indicating the possibility that the RNA sequence can be efficiently cleaved by MMP-2. To predict the cleavage rate, Michaelis-Menten kinetic parameters were calculated. As a result, the Km value was estimated to be about 120 μM and the kcat value was about 0.5 s^-1. From these values, at a physiological MMP-2 concentration (about 10 nM), the half-life of the RNA sequence was predicted to be about 3 - 4 days. This degradation rate is consistent with the time scale of tissue remodeling and is considered to be able to appropriately support cell invasion and tissue formation. With the incorporation of this sequence, the mechanical properties (persistence length) of the nanotubes decreased by about 8.7 ± 0.9%, but the self-assembly ability was maintained. Also, as a result of simulating the behavior of the nanotubes after cleavage by MMP-2 / 9, it was confirmed that the nanotubes were fragmented at the cleavage site and decomposed into shorter segments. These segments still maintain structural integrity and are predicted to be able to perform cell support functions. The formation process and structural properties of a 3D mesh-like network composed of a large number of nanotubes were simulated. The simulations were performed on a system containing up to 10^6 tiles, and it was predicted that it would take about 24 - 48 hours to form the final network structure. The network formed from the nanotubes of the WT design exhibited a uniform mesh structure with an average pore size of approximately 0.5 ± 0.1 Mm. The pore size distribution followed a log-normal distribution, with a minimum pore size of approximately 0.1 Mm and a maximum pore size of approximately 2 Mm. To evaluate the connectivity of the network, percolation analysis was performed, and it was confirmed that a continuous network was formed throughout the system, exceeding the percolation threshold at a tile concentration of 5 MM or more. To evaluate the mechanical properties of the network, virtual compression and shear tests were performed. The compression modulus was approximately 0.5 ± 0.1 kPa, which is close to the values of soft tissues (e.g., adipose tissue, approximately 0.1 - 1 kPa). The shear modulus was approximately 0.2 ± 0.05 kPa, which was approximately 40% of the compression modulus. These values strongly depended on the tile concentration, and when the concentration doubled, the modulus increased by approximately three times. Also, to evaluate the viscoelastic properties of the network, creep and stress relaxation tests were virtually performed, and the network showed distinct viscoelastic behavior, with a relaxation time in the range of approximately 10 - 100 seconds. The network formed from the nanotubes of the iSpi design exhibited a mesh structure with an average pore size of approximately 0.6 ± 0.1 Mm. The slightly larger pore size compared to the WT design is thought to be due to the shorter persistence length of the nanotubes, allowing them to bend more flexibly. Regarding the mechanical properties, the compression modulus was approximately 0.3 ± 0.08 kPa, and the shear modulus was approximately 0.12 ± 0.03 kPa, forming a softer network than the WT design. The network formed from dsOV-designed nanotubes exhibited a mesh structure with an average pore size of approximately 0.8 ± 0.15 Mm. It is considered that the pore size became the largest because the persistence length of the nanotubes was the shortest and they could bend most flexibly. Also, under the condition of high magnesium concentration (20 mM), the network structure changed significantly due to the bundle formation of nanotubes. The network after bundle formation exhibited a coarse mesh structure with an average pore size of approximately 1.5 ± 0.2 Mm, and the compressive elastic modulus increased to approximately 0.8 ± 0.15 kPa and the shear elastic modulus increased to approximately 0.3 ± 0.07 kPa. This is considered to be because the rigidity of the bundle is higher than that of a single nanotube. The network formed from the ring structure of WT-mut design exhibited characteristics significantly different from other designs. The ring structure tended to orient in the plane and formed a layered network. Within each layer, the rings partially overlapped to form a dense mesh structure with an average pore size of approximately 30 ± 5 nm. Between the layers, a cylindrical structure was formed by the stacking of rings, and these cylinders served to connect the layers. Overall, a layered-cylindrical hierarchical network structure was formed, which was similar to the basic structure of a blood vessel network. Regarding the mechanical properties, the in-plane elastic modulus (approximately 1.2 ± 0.2 kPa) was higher than the out-of-plane elastic modulus (approximately 0.3 ± 0.07 kPa), showing distinct anisotropy. The network formed from the nanotubes of Func-WT design was predicted to have basic structural characteristics similar to those of the WT design, but due to the presence of functional aptamers, the cell interaction characteristics were predicted to be significantly different. In particular, due to the presence of the RGD-mimicking aptamer, cell adhesion sites were provided throughout the network, and it was predicted that cell adhesion and invasion would be promoted. Also, due to the presence of the MMP-2 / 9 cleavage site, it was predicted that the network would be gradually degraded in response to MMP secreted by cells, promoting cell migration and tissue remodeling. As a result of simulating network formation within a space mimicking spheroids of cell size (with a diameter of approximately 20 μm), interesting spatial inhomogeneities were observed. In the central part of the spheroid, the tile concentration was low due to diffusion limitation, and a sparse network was formed. On the other hand, in the peripheral part of the spheroid, the tile concentration was high, and a dense network was formed. This inhomogeneity is an important finding for predicting behavior in the actual tissue environment. To investigate the influence of cells on network formation, simulations were also carried out in a system containing obstacles mimicking the presence of cells. As a result, it was observed that nanotubes tended to orient along the cell surface around the cells, suggesting that this might affect the cell morphology and orientation. Also, in the space between cells, it was suggested that nanotubes were oriented to bridge between cells, providing a possibility of mechanical connection between cells. To simulate the dynamic changes of the network in response to external stimuli, simulations were also carried out in a system containing multiple DNA templates under different promoter controls. For example, in a system combining the WT design under a constitutive promoter control and the dsOV design under a light-responsive promoter control, a network composed of nanotubes of the highly rigid WT design was formed in the initial stage, and then, in response to light irradiation, nanotubes of the more flexible dsOV design were formed, and it was predicted that the mechanical properties of the network would gradually change. Specifically, the compressive elastic modulus was approximately 0.5 kPa in the initial stage (0 - 12 hours), but gradually decreased and reached approximately 0.3 kPa after light irradiation (12 - 24 hours). This dynamic change is an important property for providing an optimal environment according to the developmental stage of the tissue. Molecular dynamics simulations confirmed that the RNA origami tiles of the present invention are efficiently folded and self-organized simultaneously with transcription to form nanotubes and further form a 3D mesh-like network. It was also shown that the mechanical properties (persistence length from 0.8 to 3.4 Mm) can be precisely controlled by modifying the RNA sequence, and the structure and function are maintained even when functional aptamers are incorporated. Furthermore, it was predicted that a dynamic network capable of changing its properties in response to external stimuli can be formed. These results strongly support that the RNA origami-based extracellular matrix mimicking material of the present invention is theoretically feasible and a promising approach in tissue engineering.

[0035] As optimization parameters based on the simulation results, the following are recommended: 1. Magnesium ion concentration: 6 - 10 mM (optimal balance between folding efficiency and self-organization) 2. RNA origami tile concentration: 5 - 10 MM (high efficiency of nanotube formation) 3. Linker length of functional aptamer: AAAAA (optimal balance between structural freedom and stability) 4. Number of aptamers per tile: 1 - 2 (optimal balance between folding efficiency and function) 5. GC content: adjusted according to the purpose (75% for bone / cartilage tissue, 60 - 65% for skin / muscle tissue, 50 - 55% for nerve / adipose tissue) 6. Length of double-stranded overhang: adjusted according to the purpose (0 base pairs for high rigidity, 10 base pairs for medium rigidity, 20 base pairs for low rigidity) 7. Promoter selection: adjusted according to the purpose (constitutive promoter for initial structure, light / pH / temperature-responsive promoter for responsive structure) These parameters serve as important guidelines in the actual experimental design and provide a basis for the practical application of the present invention. In particular, in the design of RNA origamitiles with mechanical and biochemical properties suitable for different tissue types, the design of dynamic networks whose properties change in response to external stimuli, and the design of functional aptamers for optimizing cell interactions, the results of this simulation provide valuable insights. As a future prospect, it is important to experimentally verify the results of this simulation and perform further detailed design optimization. In particular, confirmation of the formation and self-organization of RNA origamitiles by in vitro transcription experiments, structural analysis of nanotubes by atomic force microscopy (AFM) and transmission electron microscopy (TEM), evaluation of the mechanical properties of 3D networks by rheology measurements, and verification of biological functions by cell culture experiments are necessary. Through these experimental verifications, it is expected that the practical application of the RNA oligomer-based extracellular matrix mimetic material of the present invention will be accelerated.

Claims

1. A programmable RNA origami-based extracellular matrix mimetic material in which RNA origami tiles self-assemble to form a three-dimensional mesh-like network, wherein the RNA origami tiles are transcribed from a DNA template by RNA polymerase and fold and self-assemble simultaneously with transcription, the RNA origami tiles have a structure including an internal kissing loop and an external kissing loop, and incorporate functional aptamers including growth factor-binding aptamers, cell adhesion motifs, and / or enzymatically degradable sequences, and the mechanical properties of the RNA origami tiles are adjustable to have a persistence length in the range of 0.8 Mm to 3.4 Mm by modification of the RNA sequence. An extracellular matrix mimetic material characterized thereby.

2. A biocompatible hydrogel containing a DNA template, RNA polymerase, nucleotide precursors, and magnesium ions, wherein the DNA template encodes a plurality of RNA origami tiles having different promoters responsive to external stimuli, the hydrogel is composed of a biocompatible polymer selected from hyaluronic acid, alginic acid, polyethylene glycol (PEG), collagen, fibrin, or combinations thereof, and RNA transcribed from the DNA template after injection or transplantation folds to form RNA origami tiles, which further self-assemble to form a three-dimensional extracellular matrix mimetic structure. A hydrogel characterized thereby.

3. A method for forming a tissue engineering scaffold using the hydrogel according to Claim 2 for forming the extracellular matrix mimetic material according to Claim 1, the method comprising injecting or transplanting the hydrogel into a target site and applying an external stimulus as needed to induce the expression of specific RNA origami tiles, allowing the RNA origami tiles to self-assemble to form a three-dimensional extracellular matrix mimetic structure, and the method being characterized by being used for forming a scaffold for wound healing, cartilage regeneration, bone regeneration, nerve regeneration, promotion of vascularization, or skin regeneration.