Three-dimensional self-assembly support

Abstract

The present disclosure relates to a three-dimensional scaffold comprising a plurality of scaffold elements, each scaffold element comprising at least one amino acid peptide component and at least two nucleic acid components, the peptide component consisting of a length of amino acid wherein the at least two nucleic acid component portions of the plurality of scaffold elements are configured to mediate self-assembly of the scaffold.

Description

Technical Field

[0001] The present disclosure relates to a three-dimensional scaffold comprising a plurality of scaffold elements, each scaffold element comprising at least one peptide component and at least two nucleic acid components, the peptide portion consisting of a stretch of amino acids, wherein the at least two nucleic acid components of the plurality of scaffold elements are configured to mediate self-assembly of the scaffold. Background Art

[0002] Three-dimensional (3D) self-assembling scaffolds play a vital role in various fields of life sciences.

[0003] Scaffolds provide a structural framework that mimics the extracellular matrix (ECM) found in biological tissues. This structural support is essential for tissue engineering and regenerative medicine applications, allowing cells to adhere, proliferate, and differentiate within a 3D environment.

[0004] Many self-assembling scaffolds are made from natural or biocompatible materials, such as peptides, proteins, or polysaccharides. This biocompatibility reduces the risk of immune rejection or adverse reactions when implanted or used in contact with biological systems.

[0005] Self-assembling scaffolds offer tunable properties, including mechanical strength, porosity, degradation rate, and bioactivity. These properties can be precisely engineered to match the needs of specific tissues or applications, providing both versatility and customization.

[0006] Stents can encapsulate bioactive molecules, such as drugs, growth factors, or genetic material, and release them in a controlled manner over time. This controlled release profile improves the effectiveness and safety of treatments by maintaining optimal concentrations at the target site while minimizing systemic side effects.

[0007] Many self-assembling scaffolds can be manufactured using simple and scalable techniques, including self-assembly, molecular self-organization, or templating. This ease of fabrication facilitates large-scale production and reduces manufacturing costs, making them more accessible for commercial and clinical use.

[0008] Self-assembling scaffolds provide a 3D microenvironment that closely resembles native tissue architecture. This 3D structure allows cells to interact in a more physiologically relevant manner, thereby promoting cell-cell communication, tissue organization, and functional tissue regeneration.

[0009] Self-assembling scaffolds can be customized for a wide range of applications, including tissue engineering, drug delivery, biosensing, diagnostics, and nanotechnology. Their versatility makes them suitable for addressing diverse challenges in biomedicine and advancing research in diverse fields.

[0010] Overall, the advantages of self-assembling scaffolds have led to their widespread application and played an important role in promoting biomedical research, thereby promoting innovations in tissue engineering, drug delivery, and regenerative medicine.

[0011] Several different stents have been reported in the prior art.

[0012] Due to their inherent amphiphilic properties, peptides can self-assemble into various nanostructures and scaffolds. Peptide-based scaffolds have been utilized in tissue engineering, drug delivery, and regenerative medicine. Examples include amphiphilic peptides (Pas), which can form nanofibers, nanotubes, and other structures.

[0013] DNA origami is a technology that folds single-stranded DNA molecules into precise 3D shapes through base pairing interactions (i.e., hybridization). In addition to DNA origami, nanostructures based on DNA bricks have been developed, which are self-assembled from smaller DNA bricks and therefore do not require long scaffold chains. Despite these advances, it remains challenging to efficiently generate larger DNA nanostructures. For example, a large cube based on DNA bricks with a side length of 100 nm can be synthesized with an overall efficiency of only about 1%. This has promoted the development of Meta-DNA, which is constructed from smaller DNA nanostructure units that are assembled by pairing of staple strands. These DNA nanostructures can be used as scaffolds for various applications, such as drug delivery, biosensing, and nanoelectronics.

[0014] Proteins can self-assemble into complex 3D structures, making them useful scaffolds in biotechnology and biomedicine. Examples include virus-like particles (VLPs), which are self-assembling protein cages derived from viral capsid proteins. VLPs have been studied for drug delivery, vaccine development, and nanotechnology applications.

[0015] Hydrogels are 3D networks of hydrophilic polymers that can absorb and retain large amounts of water. They can self-assemble through various mechanisms, such as physical crosslinking, chemical crosslinking, or self-assembly of amphiphilic molecules. Due to their biocompatibility and tunable properties, hydrogels have been widely used in tissue engineering, wound healing, and drug delivery.

[0016] Nanocellulose derived from plant sources can self-assemble into nanofibrils and nanocrystals. These nanocellulose scaffolds have attracted attention in tissue engineering, wound dressings, and as drug delivery vehicles due to their biocompatibility, mechanical strength, and tunable properties.

[0017] Amphiphilic peptide molecules can self-assemble into nanofibers, and their structure and function can be precisely controlled. According to previous studies, these nanofiber scaffolds are used in tissue regeneration, neural tissue engineering, and drug delivery.

[0018] Various synthetic polymers can self-assemble into 3D structures through non-covalent interactions such as hydrogen bonding, π-π stacking, and hydrophobic interactions. These self-assembling polymer scaffolds have been explored for applications in drug delivery, tissue engineering, and nanotechnology.

[0019] These examples highlight the diverse array of self-assembling scaffolds used in the life sciences and related fields, each with unique properties and applications.

[0020] While self-assembling scaffolds offer many advantages, they also present certain limitations and disadvantages:

[0021] Controlling the degradation rate of a scaffold to match tissue regeneration can be challenging: the scaffold may degrade too quickly, resulting in insufficient support for tissue growth, or too slowly, leading to long-term inflammation or a foreign body response.

[0022] Some self-assembling scaffolds may lack sufficient mechanical strength or stiffness to withstand physiological loads, especially in load-bearing tissues. Achieving the desired mechanical properties while maintaining biocompatibility and biodegradability can be difficult.

[0023] Although many self-assembling scaffolds are made of biocompatible materials, they can still trigger an immune response in some individuals, leading to inflammation, fibrosis, or rejection. Immune responses can impair the function and integration of scaffolds within host tissues.

[0024] Fabricating self-assembling scaffolds with precise control over their structure and properties can be complex and require sophisticated techniques. Ensuring the reproducibility and scalability of the manufacturing process can pose challenges, particularly for clinical translation and commercialization.

[0025] While bioactive molecules can be incorporated into some scaffolds to enhance functionality, achieving sustained and controlled release of these molecules can be difficult. Maintaining bioactivity over time and ensuring proper spatial distribution within the scaffold are also challenging.

[0026] Introducing new biomaterials and scaffolds into clinical practice requires a rigorous regulatory approval process to ensure safety and efficacy. Meeting regulatory standards for biocompatibility, sterility, and clinical performance increases the time and cost of developing and commercializing new scaffolds.

[0027] Ensuring proper integration of scaffolds with host tissues and promoting functional regeneration remains a major challenge. Scaffold design must consider factors such as cell adhesion, migration, vascularization, and innervation to support tissue remodeling and effectively restore tissue function.

[0028] Developing and manufacturing self-assembling scaffolds can be expensive using advanced biomaterials and fabrication techniques. High production costs may limit their widespread application, especially in resource-limited settings or large-scale applications.

[0029] Therefore, there is a need in the art to overcome the aforementioned disadvantages. The three-dimensional self-assembling scaffolds according to embodiments of the present disclosure do overcome these disadvantages, thereby retaining the advantages associated with such scaffolds. Summary of the Invention

[0030] In a first aspect, the present disclosure relates to a three-dimensional scaffold comprising a plurality of scaffold elements, each scaffold element comprising at least one peptide component and at least two nucleic acid components, wherein the peptide portion is composed of a stretch of amino acids, wherein the at least two nucleic acid components of the plurality of scaffold elements are configured to mediate self-assembly of the scaffold.

[0031] In a second aspect, the present disclosure relates to a method for producing a three-dimensional scaffold, the method comprising the steps of: a. Providing a plurality of scaffold elements, the scaffold element comprising at least one peptide component and at least two nucleic acid components; b. wherein, optionally, at least one of the scaffold elements further comprises a binding element, the binding element being selected from a nucleic acid-based binding region and / or a peptide-based binding region; c. placing the scaffold elements in a solution, whereby self-assembly of the scaffold elements occurs, and d. Obtain the prepared three-dimensional scaffold.

[0032] In a third aspect, the present disclosure relates to a three-dimensional scaffold produced by the disclosed method.

[0033] In a fourth aspect, the present disclosure relates to uses of the disclosed three-dimensional scaffolds, selected from the group consisting of uses in bioimaging and labeling, uses in templates for materials synthesis, uses in molecular sensing, uses in diagnostic tools, uses in molecular robotics and computing, uses in synthetic biology, uses in bottom-up nanofabrication, uses in nanoscale devices, uses in bioprocessing, and uses in bioprinting, and combinations thereof.

[0034] In a fifth aspect, the present disclosure relates to a three-dimensional scaffold disclosed below for use as a pharmaceutical, such as in tissue and / or cell repair, tissue and / or cell engineering, drug delivery, wound treatment, bone reconstruction, construction and partial construction of artificial organs, and combinations thereof.

[0035] In a sixth aspect, the present disclosure relates to a kit comprising a three-dimensional scaffold disclosed below, further comprising an item selected from the group consisting of a buffer, a package insert, an applicator, a dosing device, a mixing device, a manual, a device for inducing polymerization, a dye, and a hydrogel matrix, and combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 - Schematic diagram of a three-dimensional self-assembling scaffold according to an embodiment of the present disclosure. The three-dimensional scaffold comprises a plurality of scaffold elements (101), which may be the same or different from each other. Each scaffold element comprises at least one peptide component (102) and at least two nucleic acid components (103, 104), wherein the peptide component is composed of a stretch of amino acids, wherein the at least two nucleic acid components (103, 104) of the plurality of scaffold elements (101) are configured to mediate self-assembly of the scaffold (100).

[0037] Figure 2 - Schematic diagram of a scaffold element (101). The element may include an internal nucleic acid-based binding region (105) and / or an internal peptide-based binding region (106) that facilitates further binding to a target selected from the group consisting of nucleic acids, peptides, proteins, inorganic molecules (e.g., toxins or radionuclides), organic molecules (e.g., microplastics, dyes or fluorescent agents), sugars and / or lipids. The scaffold element (101) may also include one or more PEVK motifs in its peptide component (102).

[0038] Figure 3 Another schematic diagram of a three-dimensional self-assembling scaffold according to an embodiment of the present disclosure. This diagram depicts the interaction of different elements. In one embodiment, at least two nucleic acid components (103, 104) located at the ends of a scaffold element (101a) comprise nucleic acid regions (e.g., A, B) that specifically hybridize with nucleic acid regions (e.g., A', B') on other scaffold elements (101b), thereby promoting directed self-assembly. The at least two nucleic acid components (103, 104) can be present in multiple copies at the ends of the scaffold elements (101a, 101b).

[0039] Figure 4- Schematic representation of different scaffold elements (101). Depending on the envisaged three-dimensional structure of the scaffold, the at least two nucleic acid components (103, 103a, 103b, ..., 104, 104a, 104b, ...) of the scaffold element, in one embodiment located at its ends, can be designed for different functional purposes. Hybridization strength and specificity can be enhanced by the composition ("barcode") and / or length of the complementary nucleic acid sequences and / or by the number of copies (1, 2, 3, ...) of the nucleic acid components.

[0040] Figure 5 - Schematic representation of complementary binding elements. For example, nucleic acid sequence ("barcode") "A" can hybridize to barcode A'. Alternatively, A can be affinity partner A that binds to affinity partner A'.

[0041] Figure 6 - Another schematic diagram of a three-dimensional self-assembling scaffold according to an embodiment of the present disclosure. Different scaffold elements may additionally include internal nucleic acid-based binding regions (105) and / or internal peptide-based binding regions (106) that facilitate further binding to a target selected from the group consisting of nucleic acids, peptides, proteins, inorganic molecules (e.g., toxins or radionuclides), organic molecules (e.g., microplastics, dyes or fluorescent agents), sugars and / or lipids. The figure depicts an embodiment of an aptamer (108) comprising a region that hybridizes to the internal nucleic acid-based binding region (105a) and further binds to a protein target. Also depicted is an embodiment of a cross-linked nucleic acid component (109) comprising a region that hybridizes to the internal nucleic acid-based binding region (105b) of two different scaffold elements.

[0042] Figure 7 Another schematic diagram of a three-dimensional self-assembling scaffold according to an embodiment of the present disclosure. This figure depicts a general three-dimensional structure and an example in which different scaffold elements (101) include internal peptide-based binding regions (106) that facilitate further binding to a target selected from the group consisting of nucleic acids, peptides, proteins, inorganic molecules (e.g., toxins or radionuclides), organic molecules (e.g., microplastics, dyes or fluorescent agents), sugars and / or lipids.

[0043] Figure 8- Schematic diagrams of multilayer three-dimensional self-assembling scaffolds (100a-100f) according to embodiments of the present disclosure. Combinations of multilayer three-dimensional self-assembling scaffolds according to the present disclosure may be useful, particularly in the context of tissue engineering and / or complex filtration systems. This can be achieved by combining three-dimensional self-assembling scaffolds with certain physical properties (i.e., mesh structure, pore size, PDI) and / or different biochemical properties, and incorporating specific binding moieties of other three-dimensional self-assembling scaffolds at their interfaces.

[0044] Figure 9 - Schematic diagram of a "corner" of a three-dimensional self-assembling scaffold according to embodiments of the present disclosure. The at least two nucleic acid components (103, 104) at the ends of the scaffold elements (101a-f) each comprise at least two different nucleic acid regions (e.g., A, A') that specifically hybridize with nucleic acid regions (e.g., A', A) on other scaffold elements (101a-f), thereby promoting directed self-assembly.

[0045] Figure 10 - Schematic diagram of a "corner" of a three-dimensional self-assembling scaffold according to an embodiment of the present disclosure. Figure 9 The at least two nucleic acid components (103, 104) at the ends of the scaffold element (101a-f) each comprise at least two identical nucleic acid regions (e.g., A, A or A', A'), which specifically hybridize with nucleic acid regions on other scaffold elements (101a-f), thereby promoting directed self-assembly.

[0046] Figure 11 - Schematic diagram of a typical scaffold element (101) according to the present disclosure. The scaffold element (101) comprises a nucleic acid region at each end, which is two copies of SEQ ID NO:37 or SEQ ID NO:38, and in one embodiment, is interspersed with a 5-nucleotide nonsense nucleic acid region (i.e., CCCCC).

[0047] Figure 12 - Schematic representation of the "expandability" of a scaffold element. In one embodiment the scaffold element, in one embodiment the peptide component of the scaffold element, can be expanded at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold or at least 1000-fold. Reference signs of the drawings:

[0048] 100 - Three-dimensional self-assembling scaffold (In some figures, elements 100a-100f are used to depict different three-dimensional self-assembling scaffolds)

[0049] 101 - scaffold element, suitable for forming a three-dimensional self-assembling scaffold (in some figures, elements 101a, 101b, etc. are used to depict different scaffold elements)

[0050] 102-Peptide component of the scaffold element

[0051] 103 - first nucleic acid component of the scaffold element (in some figures, elements 103a, 103b, etc. are used to depict different first nucleic acid components and variants); Figure 4 The “1, 2, 3, 4, . . . ” in the first nucleic acid component refers to multiple copies of the same or different first nucleic acid components.

[0052] 104 - Second nucleic acid component of the scaffold element (in some figures, elements 104a, 104b, etc. are used to depict different second nucleic acid components and variants); Figure 4 The “1, 2, 3, 4, . . . ” in the second nucleic acid component refers to multiple copies of the same or different second nucleic acid components.

[0053] 105 - An internal nucleic acid based binding region that is distinct from elements (103, 104) and hybridizes to the nucleic acid target.

[0054] 106-An internal peptide-based binding region that binds a target selected from the group consisting of nucleic acids, peptides, proteins, inorganic molecules (e.g., toxins or radionuclides), organic molecules (e.g., microplastics, dyes or fluorescent agents), sugars and / or lipids.

[0055] 107-amino acid motif consisting of proline (P), glutamic acid (E), valine (V), and lysine (K) (PEVK)

[0056] 108 - Embodiments of molecules that can hybridize to an internal nucleic acid-based binding region (105); The figure depicts an aptamer that allows binding to a target selected from the group consisting of nucleic acids, peptides, proteins, inorganic molecules (e.g., toxins or radionuclides), organic molecules (e.g., microplastics, dyes or fluorescent agents), sugars and / or lipids.

[0057] 109 - Example of a molecule that can hybridize to the internal nucleic acid based binding region (105); depicted as another nucleic acid that forms an additional intramolecular bridge with another scaffold element.

[0058] A, A' - Schematic representation of substantially complementary nucleic acid sequences capable of specifically hybridizing to each other (B, B'; C, C' etc.). DETAILED DESCRIPTION

[0059] The present disclosure relates to a three-dimensional scaffold comprising a plurality of scaffold elements, each of which comprises at least one peptide component and at least two nucleic acid components, wherein the peptide component is composed of a stretch of amino acids, wherein the at least two nucleic acid components of the plurality of scaffold elements are configured to mediate self-assembly of the scaffold.

[0060] Such a three-dimensional scaffold can also be called a "hybrid scaffold" because it contains both peptide and nucleic acid elements. Therefore, it combines the best of both worlds: the versatility, flexibility, and strength of peptide backbones with the programmability and self-assembly ability of nucleic acids.

[0061] Peptide components

[0062] The peptide component serves a variety of purposes in terms of the rigidity, flexibility, elasticity and size of the three-dimensional scaffold, which affect the design and selection of peptides. Therefore, the at least one peptide component comprises at least one characteristic selected from the group consisting of: a. A longitudinal tensile strength of at least 350 MPa, at least 375 MPa, at least 400 MPa, at least 425 MPa, at least 450 MPa, at least 475 MPa, at least 500 MPa, or at least 550 MPa. In one embodiment, the longitudinal tensile strength is 800 MPa or less, 700 MPa or less, 600 MPa or less, 590 MPa or less, 580 MPa or less, or 560 MPa or less. In one embodiment, the longitudinal tensile strength is from 350 MPa to 800 MPa, from 400 MPa to 700 MPa, from 500 MPa to 600 MPa, or from 525 MPa to 575 MPa. b. A tensile modulus of at least 3.5 GPa, at least 3.75 GPa, at least 4.0 GPa, at least 4.25 GPa, at least 4.5 GPa, at least 4.75 GPa, at least 5.0 GPa, or at least 5.25 GPa. In one embodiment, the tensile modulus is 10.0 GPa or less, 9.0 GPa or less, 8.0 GPa or less, 7.0 GPa or less, or 6.0 GPa or less. In one embodiment, the tensile modulus is from 3.5 GPa to 10.0 GPa, from 4.0 GPa to 9.0 GPa, from 4.5 GPa to 8.0 GPa, from 5.0 GPa to 7.0 GPa, or from 5.5 GPa to 6.0 GPa. c. A ductility of at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%. In one embodiment, the ductility is less than 100%, less than 95%, less than 90%, or less than 85%. In one embodiment, the ductility is between 45% and 100%, between 50% and 95%, between 55% and 90%, between 60% and 85%, or between 65% and 80%. d. At least 120Mj / m 3 , at least 130Mj / m 3 , at least 140Mj / m 3 , at least 150Mj / m 3 , at least 160Mj / m 3 , at least 170Mj / m 3 , at least 180Mj / m 3 or at least 190Mj / m 3 In one embodiment, the toughness is 250Mj / m 3 Below, 240Mj / m 3 Below, 230Mj / m 3 Below, 220Mj / m 3 Below, or 210Mj / m 3 In one embodiment, the toughness is 120 Mj / m 3 Up to 250Mj / m 3 、130Mj / m 3 Up to 240Mj / m 3 、140Mj / m 3 Up to 230Mj / m 3 、150Mj / m 3 Up to 220Mj / m 3 , or 160Mj / m 3 Up to 210Mj / m 3 . e. The length of the peptide component (102) is at least 0.5 μm, at least 0.6 μm, at least 0.7 μm, at least 0.8 μm, at least 0.9 μm, at least 1.0 μm, at least 1.1 μm, at least 1.2 μm, at least 1.3 μm, or at least 1.5 μm. In one embodiment, the length of the peptide component (102) is 5 μm or less, 4 μm or less, 3 μm or less, 2 μm or less, or 1.8 μm or less. In one embodiment, the length of the peptide component (102) is 0.5 μm to 5 μm, 0.6 μm to 4 μm, 0.7 μm to 3 μm, 0.8 μm to 2 μm, or 1.0 μm to 1.5 μm. f. comprises at least 3000 amino acids, at least 5000 amino acids, at least 10000 amino acids, at least 15000 amino acids, at least 20000 amino acids, at least 21000 amino acids, at least 22000 amino acids, at least 22500 amino acids, at least 22700 amino acids, at least 22720 amino acids, at least 22800 amino acids, or at least 22900 amino acids. In one embodiment, the peptide component (102) comprises 50000 amino acids or less, 45000 amino acids or less, 40000 amino acids or less, 38000 amino acids or less, or 37000 amino acids or less. In one embodiment, the peptide component (102) comprises 3000 to 50000 amino acids, 5000 to 45000 amino acids, 10000 to 40000 amino acids, or 20000 to 38000 amino acids.

[0063] Obviously, the molecular weight of the peptide component does not necessarily need to be 2.5 MDa; this value is intended only to enable comparison of different structural proteins, and therefore should be understood as determining the size of the physical parameters mentioned using common test methods. In some embodiments, the peptide component may contain two or more copies and / or partial copies of the amino acid sequence to achieve the desired length. In some embodiments, the peptide component may contain two or more copies and / or partial copies of a peptide selected from SEQ ID NOs: 1-36, as well as variants and isoforms thereof.

[0064] There are naturally occurring structural proteins that, due to their role in nature, already possess the necessary properties that are also desired in the disclosed three-dimensional scaffolds.

[0065] Such structural proteins can be, for example, cytoskeletal proteins, which naturally play a key role in maintaining the structural integrity of cells, particularly in cell shape, stability, and membrane organization, proteins of the extracellular matrix, microfilament family proteins, and / or the environment of a portion of a muscle fiber. A group of such naturally occurring structural proteins consists of nebulin, obscurin, dystrophin, and titin, and combinations thereof.

[0066] Thus, in one embodiment, the at least one peptide component (102) is selected from the group consisting of chaperone, obscurin, dystrophin, and titin, and combinations thereof.

[0067] In one embodiment, the protein of SEQ ID NO: 1-36 is preferred. In one embodiment, the peptide component comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1 to 36, as well as variants, fragments, and isoforms thereof. In one embodiment, the protein is titin (SEQ ID NO: 1-13) or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.9% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1 to 13, as well as variants, fragments, and isoforms thereof.

[0068] For the correct function of the scaffold, the peptide component (102) must not contain any peptide-nucleic acid variants (PNA). Therefore, in one embodiment, the peptide component (102) consists only of a stretch of amino acids connected by peptide bonds. Therefore, in one embodiment, the peptide component (102) does not contain any PNA.

[0069] In one embodiment, the peptide component comprises at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, or at least 400 Ig-like domains.

[0070] In one embodiment, the peptide component comprises at least 50, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 125, at least 130, at least 135 type I fibronectin type III domains.

[0071] In one embodiment, the peptide component comprises at least 50, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115 type II immunoglobulin domains.

[0072] In one embodiment, the peptide component comprises at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24 Proline-Glutamic Acid-Valine-Lysine (PEVK) motifs.

[0073] In one embodiment, the peptide component (102) may further comprise at least one peptide-based binding region (106). The at least one peptide-based binding region (106) may be selected from the group consisting of antigen-binding peptides (such as antibodies, Fab fragments and / or svFc fragments), peptide aptamers, DARPins (designed ankyrin repeat proteins), affimers, nanobodies, click-chemistry-based ligands, polymeric conjugates, affinity tags (HA, Myc, FLAG), protein A, protein G, streptavidin and aptazymes, helix-turn-helix (HTH) domains, zinc finger domains, leucine zipper domains, basic helix-loop-helix (bHLH) domains, homeotic domains, AT-hook motifs, arginine-glycine-rich (RGG) domains, RNA recognition motifs (RRMs), cys-2-his-2-zinc fingers (C2H2), and polycomb repressive complex (PRC1) ring finger domains, and combinations thereof.

[0074] Nucleic acid components

[0075] The three-dimensional scaffold of the present disclosure comprises a plurality of scaffold elements, each of which may comprise at least two nucleic acid components.

[0076] The at least two nucleic acid components include a first nucleic acid component and a second nucleic acid component, the second nucleic acid component being different from the first nucleic acid component, wherein the first nucleic acid component and the second nucleic acid component are each configured to hybridize to a different target.

[0077] In one embodiment, the nucleic acid component of each scaffold element is configured not to bind to itself, i.e., the nucleic acid component of a first scaffold element (e.g., 101a) will not bind to the nucleic acid component of a second scaffold element (e.g., 101b). However, in one embodiment, the nucleic acid component of a first scaffold element (e.g., 101a) may bind to the nucleic acid component of a second scaffold element (e.g., 101b).

[0078] In one embodiment, the nucleic acid component is located at each end of the scaffold element, where "end" can be the most distal portion of each scaffold element in one embodiment, but in some embodiments also includes configurations where the nucleic acid component is located within 10, 20, 30, 40, or 50 amino acids and / or 10, 20, 30, 40, or 50 nucleic acids of one of the ends of the scaffold element.

[0079] Furthermore, within each of the at least two nucleic acid components, different hybridization regions can be defined that allow specific hybridization to different targets. For example, a nucleic acid component may comprise a nucleic acid sequence A that is capable of hybridizing to a nucleic acid sequence A', but may additionally comprise a nucleic acid sequence B that is capable of hybridizing to a nucleic acid sequence B', and so on.

[0080] In one embodiment, the at least two nucleic acid components can be encoded to specifically hybridize to at least one nucleic acid moiety in the nucleic acid component of another scaffold element, thereby mediating self-assembly of the scaffold.

[0081] Generally, the nucleic acid components are designed to facilitate a three-dimensional structure. Each "corner" of the three-dimensional scaffold requires providing at least two specific contacts to at least five other different scaffold elements (see Figure 9 ). This can be achieved at a minimum by at least two different nucleic acid sequences (A, A') per nucleic acid component (i.e. per "end" of each scaffold element), wherein A hybridizes to A'. However, in order to facilitate self-assembly in a more controlled manner and / or more complex structures, different nucleic acid components can comprise more than two sequences and / or more than two copies of the same or different sequences.

[0082] In one embodiment, the nucleic acid sequences to be used for A, A', B, B', etc. should be unique sequences that are not present in the target tissue or subject. This can be achieved by synthetic sequences and / or by using naturally occurring unique sequences, such as a part of the nucleic acid sequence of the human hemoglobin subunit beta (see SEQ ID NO: 37).

[0083] Thus, in one embodiment, each of the at least two nucleic acid components can comprise two different sequences A and A', or one sequence pair, that hybridize to each other. In further embodiments, each of the at least two nucleic acid components can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different sequence pairs that hybridize to each other.

[0084] In order to facilitate specific hybridization, each of the sequences (i.e. A, A', B, B', etc.) comprises at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides. Fewer nucleotides result in non-specific hybridization, and more nucleotides generally do not improve hybridization, but are more difficult to generate. However, in special cases, longer sequences can be incorporated for particular applications.

[0085] In another embodiment, the at least two nucleic acid components are covalently conjugated to the peptide component of the scaffold element. In another embodiment, the at least two nucleic acid components are indirectly attached to the peptide component of the scaffold element by affinity interactions (e.g., aptamer-based, nanobody-based, streptavidin-biotin).

[0086] In another embodiment, the scaffold element can comprise at least one additional nucleic acid-based binding region. In comparison to the at least two nucleic acid components, the at least one additional nucleic acid-based binding region is located more proximal to the middle of the scaffold element. In one embodiment, the peptide component of the scaffold element can be enclosed on both sides thereof, thereby essentially dividing the peptide component into two parts.

[0087] In one embodiment, the at least two nucleic acid components and / or at least one nucleic acid-based binding region are selected from the group consisting of deoxyribonucleic acid strands (DNA), L-DNA, D-DNA, PT-DNA, ribonucleic acid strands (RNA), xenonucleic acid strands (XNA), peptide nucleic acid strands (PNA), locked nucleic acid strands (LNA), and morpholino nucleic acid strands (MNA), and combinations thereof.

[0088] In another embodiment, the at least two nucleic acid components and / or at least one nucleic acid-based binding region can comprise at least one structural element selected from the group consisting of nucleic acid origami elements, such as DNA tiles, DNA bricks, RNA tiles, RNA bricks, XNA tiles, XNA bricks, PNA tiles, PNA bricks, LNA tiles, LNA bricks, MNA tiles, and MNA bricks, and combinations thereof; and / or a binding element selected from the group consisting of aptamers, DNAzymes (deoxyribozymes), riboswitches, and aptazymes, and combinations thereof.

[0089] In one embodiment of the present disclosure, the nucleic acid elements are “programmable”. That is, they are designed to carry a certain “barcode”, i.e., a specific nucleic acid sequence that allows for controlling and modulating the self-assembly of the scaffold. They also allow for fine-tuning the features of the scaffold, such as mesh size, polydispersity index (PDI), overall strength of the scaffold, water binding capacity, binding capacity to target molecules, cross-linking, etc.

[0090] In one embodiment, the present disclosure also encompasses expression vectors for producing the scaffold element in bacteria and / or yeast. Such vectors typically comprise: • A promoter sequence, typically derived from a strong and constitutively active promoter such as the CMV (cytomegalovirus) promoter or the SV40 (simian virus 40) promoter, drives transcription of the gene of interest. The promoter initiates the transcription process by recruiting RNA polymerase and other transcription factors to the transcription start site of the gene. • The expression vector contains a gene for encoding the scaffold element (101) (or at least a part thereof). The gene is typically inserted into the vector using specific restriction enzyme sites or other cloning techniques. • A selective marker gene, such as an antibiotic resistance gene (e.g., ampicillin resistance gene, kanamycin resistance gene) or a reporter gene (e.g., GFP, beta-galactosidase), is included in the vector to facilitate selection and identification of host cells that have successfully taken up the vector. Cells that have incorporated the vector can be selected by culturing them in media containing the corresponding selection agent. • A replication origin is a DNA sequence that allows the vector to replicate independently of the chromosomal DNA of the host organism. It ensures that the vector is replicated and maintained in the host cell during cell division. • A polyadenylation signal sequence, typically derived from bovine growth hormone (BGH) or SV40, is included downstream of the gene of interest. This sequence directs the addition of a poly-A tail to the mRNA transcript, thereby stabilizing the mRNA and increasing its translation efficiency. • A multiple cloning site, also known as a polylinker or restriction site, is a region within the vector that contains multiple unique restriction enzyme recognition sites. These sites allow for the insertion of the gene of interest into specific locations within the vector, facilitating cloning and manipulation of the vector. • Optionally, the expression vector can include additional enhancer elements or regulatory sequences to enhance the level of gene expression or tissue-specific expression patterns.

[0091] In one embodiment, the present disclosure also encompasses microorganisms, i.e., bacteria and / or yeast, that comprise such a vector stably and / or transiently incorporated.

[0092] Three-dimensional scaffold

[0093] In one embodiment, the three-dimensional scaffold can be further characterized by at least one of the following features: a. having a peptide content of at least 1 wt%, at least 2 wt%, at least 5 wt%, at least 10 wt%, at least 20 wt%, or at least 30 wt%. In one embodiment, having a peptide content of less than 90 wt%, less than 85 wt%, less than 80 wt%, less than 75 wt%, less than 70 wt%, less than 65 wt%, or less than 60 wt%. In one embodiment, having 1% to 40% (w / v), 5% to 35% (w / v), 8% to 30% (w / v), 10% to 25% (w / v), or 15% to 20% (w / v); and / or b. having a nucleic acid content of at least 0.01%, at least 0.02%, at least 0.05%, at least 0.1%, at least 0.5%, or at least 1% by weight. In one embodiment, the nucleic acid content is 40% or less, 35% or less, 30% or less, 28% or less, 25% or less, 22% or less, or 20% or less by weight. In one embodiment, the nucleic acid content is 0.01% to 30% (w / v), 0.05% to 20% (w / v), 0.1% to 15% (w / v), 0.5% to 10% (w / v), or 1% to 5% (w / v). c. having a water content of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% by volume. In one embodiment, having a water content of 99% or less, 98% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, or 65% or less by volume. In one embodiment, having a water content of 10% to 99% by volume, or 20% to 98% by volume, 30% to 95% by volume, 40% to 90% by volume, 50% to 85% by volume, or 60% to 80% by volume. d. having a programmable 3-D pattern, wherein the nucleic acid component and / or the peptide component is designed to form a specific 3-D pattern based on nucleic acid-nucleic acid, protein-protein and / or nucleic acid-protein interactions, wherein the characteristics of the 3-D pattern are selected from: Porous networks: Porosity is a key feature for nutrient and waste exchange, mimicking the porous structure of the natural ECM. The size, shape, and distribution of pores can be customized to match the needs of different cell types and tissues. Porous scaffolds can be created using techniques such as gas foaming, salt leaching, freeze drying, and electrospinning. Fiber networks: Electrospinning is a popular technique for creating scaffolds composed of intertwined fibers, mimicking the fibrous components of the ECM. The diameter of the fibers can vary from nanometers to micrometers, and The density and orientation of the fibers can be controlled to influence cell behavior. Layered structures: Using techniques such as 3D printing (bioprinting) or layer-by-layer assembly, scaffolds can be fabricated with precise layered structures that mimic the layered organization of tissues such as skin or cartilage. Honeycomb pattern: A honeycomb pattern characterized by regular hexagonal pores is beneficial for certain types of tissue engineering because it can enhance cell seeding and nutrient diffusion due to high porosity and uniform pore distribution. Spheroid and ellipsoid structures: Spheroid or ellipsoid structures within the scaffold are important for the culture of stem cells or tissues. Spheroid formation can be particularly useful, which is important in organoid development and cancer research. Vascular networks: Creating scaffolds with embedded vascular-like channels is particularly useful for tissues with high metabolic demands. These patterns ensure the transport of nutrients and oxygen throughout the scaffold, thereby facilitating the survival of embedded cells. Gradient structures: Some tissues require gradients of biochemical cues or physical properties (e.g., stiffness) across the scaffold to guide tissue formation. Advanced manufacturing techniques can create scaffolds with spatial gradients that mimic these natural tissue characteristics. e. is a programmable self-assembling hydrogel, in one embodiment, having a water content of at least 30% (w / v), at least 40% (w / v), at least 50% (w / v), at least 60% (w / v), at least 70% (w / v), at least 80% (w / v), at least 90% (w / v), at least 95% (w / v), or at least 99% (w / v), f. Its filling is at least 0.001μm 3 , at least 1μm 3 , at least 1000μm 3 , at least 0.001mm 3 , at least 1mm 3 , at least 1000mm 3 , at least 1cm 3 , at least 1000cm 3 or at least 0.0001m 3 The volume, g. which consists of at least 10, at least 100, at least 1000, at least 10,000 or at least 100,000 stent elements, h. embedded in a hydrogel or polymer matrix that can be stretched at least 2 times, at least 5 times, at least 10 times, at least 100 times, or at least 1000 times; i. Biodegradable in less than 1 year, less than 6 months, less than 5 months, less than 4 months, less than 3 months, less than 2 months, or less than 1 month.

[0094] In one embodiment, the present disclosure relates to a method for producing a three-dimensional scaffold, the method comprising the steps of: a. providing a plurality of scaffold elements (101), the scaffold element (101) comprising at least one peptide component (102) and at least two nucleic acid components (103, 104); b. wherein, optionally, at least one of the scaffold elements (101) further comprises a binding element selected from a nucleic acid-based binding region (105) and / or a peptide-based binding region (106); c. placing the scaffold element in a solution, whereby self-assembly of the scaffold element (101) occurs, and d. Obtain the prepared three-dimensional scaffold.

[0095] In one embodiment, the present disclosure relates to a three-dimensional scaffold produced by the method disclosed above.

[0096] The three-dimensional scaffolds of the present disclosure are suitable for a large number of uses, such as uses selected from the following: uses in biological imaging and labeling, uses in templates for material synthesis, uses in molecular sensing, uses in diagnostic tools, uses in molecular robotics and computing, uses in synthetic biology, uses in bottom-up nanofabrication, uses in nanoscale devices, uses in bioprocessing and uses in bioprinting, and combinations thereof.

[0097] Bioimaging and labeling involve techniques for visualizing and tracking biomolecules, cells, or tissues within living organisms or in vitro systems, or such as tissue sections, liquid biopsies, or lysates. These techniques can be used to understand biological processes, diagnose diseases, and develop treatments. The self-assembling scaffolds disclosed herein can be used to enhance bioimaging and labeling techniques. The self-assembling scaffolds disclosed herein can spontaneously organize into ordered arrangements and can provide a framework for precisely positioning and orienting imaging or labeling agents, thereby improving their efficiency and specificity in biological systems.

[0098] In the context of bioimaging and labeling, self-assembling scaffolds can be used for a variety of purposes: Spatial organization: The self-assembling scaffolds of the present invention can organize imaging or labeled reagents into specific patterns or arrays, thereby allowing precise spatial control of their distribution within biological samples. This spatial organization enables high-resolution visualization of molecular interactions, cell structures, or tissue architectures. Similarly, the self-assembling scaffolds of the present invention can organize target analytes into regular arrays, which can facilitate their detection. For example, in the context of multiple (100 analytes), high-weight (100-1000 analytes), or ultra-high-weight (10,000 analytes), the self-assembling scaffolds of the present invention can be used to generate regular 3D arrays that position analytes (e.g., proteins or nucleic acids) in a flow cell, thereby utilizing the entire volume of the flow cell while ensuring the ideal spacing of the target analytes relative to the resolution of the readout. For example, such an array can be used for large-scale parallel sequencing of nucleic acid targets using microscope-based readout, and the targets can be positioned to have a distance of about 240-300 nm in the XY direction and about 700-1000 nm in the Z direction. In another embodiment, the analyte can be a protein, and the readout can be based on single-molecule positioning, using, for example, DNA-PAINT or another scintillation-based technology (e.g., as disclosed in European patent application No. 23192184.2 and / or WO 2022 / 242887 A1, the entire contents of which are incorporated herein by reference), and the target-to-target distance can be in the range of a few nanometers, e.g., 5-10 nm, 10-20 nm, 10-100 nm. In another embodiment, the self-assembling scaffold of the present disclosure further includes a nanoarray based on DNA nanostructures as described in WO 2022 / 207832 A1 (the entire contents of which are incorporated herein by reference), which is configured to capture analytes in certain capture bands. This embodiment is capable of capturing a large number of analytes and reading out a large number of analytes. Targeting and delivery: The self-assembling scaffolds of the present disclosure can be designed to target specific biological structures or cells. By incorporating targeting ligands or antibodies onto the scaffold surface, imaging or labeling agents can be selectively delivered to desired locations within the body, thereby enhancing imaging specificity and reducing off-target effects. Signal amplification: The self-assembling scaffolds of the present disclosure can be engineered to amplify imaging signals, thereby increasing the sensitivity of detection. For example, compared to freely diffusing reagents, the attached self-assembling nanoparticles can Imaging agents can generate enhanced signals, allowing for the detection of low-abundance biomolecules or cells. Biocompatibility and stability: The self-assembling scaffolds disclosed herein can be designed to be biocompatible and stable in biological environments, minimizing cytotoxicity and degradation. This ensures the long-term viability of labeled cells or tissues in longitudinal imaging studies.

[0099] The self-assembling scaffolds disclosed herein can be used in the creation of new materials with desired properties through chemical, physical, or biological methods. The self-assembling scaffolds disclosed herein can be used in materials synthesis to engineer advanced materials with customized structures and functions. Here are some ways in which self-assembling scaffolds can enhance materials synthesis: Controlled assembly: The self-assembling scaffolds disclosed herein enable precise control of material assembly at the nanoscale. By designing the molecular structure and interactions of the scaffold components, researchers can determine the arrangement of the building blocks during synthesis. This control allows the creation of materials with specialized nanostructures, such as nanofibers, nanoparticles, or nanosheets, that are difficult to achieve using conventional methods. Hierarchical organization: The self-assembling scaffolds disclosed herein facilitate hierarchical organization in material synthesis, where structures are constructed from multiple organizational levels. By adjusting the properties of the scaffold components (such as their size, shape, and surface chemistry), researchers can induce hierarchical self-assembly to produce composite materials with hierarchical structures. These materials often exhibit unique properties, such as enhanced mechanical strength, conductivity, or responsiveness to external stimuli. Functionalization: The self-assembling scaffolds disclosed herein provide a versatile platform for functionalizing materials with diverse properties and functions. Functional molecules (such as polymers, nanoparticles, or biomolecules) can be incorporated into the scaffold structure during synthesis, allowing for the introduction of specific properties such as biocompatibility, catalytic activity, or optical responsiveness. This functionalization enhances the versatility and utility of the synthesized materials in a variety of applications, including biomedicine, electronics, and catalysis. Scalability and Reproducibility: The self-assembling scaffolds disclosed herein provide a scalable and reproducible approach for materials synthesis. Unlike traditional synthesis methods that rely on complex reaction conditions or expensive equipment, the self-assembly process is generally simple, cost-effective, and amenable to large-scale production. This scalability and reproducibility are crucial for translating materials developed in the laboratory into practical applications, such as industrial manufacturing or biomedical devices. Dynamic Responsiveness: The self-assembling scaffolds disclosed herein can exhibit dynamic responsiveness to external stimuli, allowing for reversible changes in material properties or structure. By incorporating stimuli-responsive components (such as temperature-sensitive polymers or pH-responsive surfactants) into the scaffold design, researchers can engineer materials that undergo controlled structural or phase transitions in response to environmental cues. This dynamic behavior enables the development of smart materials with tunable properties for applications in sensing, drug delivery, and adaptive materials.

[0100] Self-assembling scaffolds also offer significant advantages in the field of molecular sensing and diagnostic tools, improving sensitivity, specificity, and versatility. Examples include: Enhanced signal amplification: The self-assembling scaffolds of the present disclosure can facilitate signal amplification in molecular sensing assays. By incorporating multiple sensing elements or reporter molecules onto the scaffold surface, researchers can amplify the detection signal, enabling sensitive detection of target molecules even at low concentrations. This enhanced sensitivity is crucial for diagnostic applications, where early detection of biomarkers or pathogens is essential for disease diagnosis and monitoring. Multiplexed Detection: The self-assembling scaffolds disclosed herein enable multiplexed detection of multiple analytes in a single assay. By immobilizing different sensing elements or probes to different regions of the scaffold, researchers can simultaneously detect multiple targets within complex samples. This multiplexing capability increases the throughput and efficiency of diagnostic assays, allowing for comprehensive analysis of biomolecular profiles or disease markers in biological samples. Spatial Organization and Immobilization: The self-assembling scaffolds disclosed herein provide a platform for spatially organizing and immobilizing sensing elements with precise control. By designing the scaffold structure and surface chemistry, researchers can immobilize sensing probes in predefined positions or orientations, thereby increasing their accessibility to target molecules. This spatial organization improves the specificity and efficiency of molecular recognition, thereby reducing nonspecific binding and background noise in diagnostic assays. Dynamic Sensing Platform: The self-assembling scaffolds disclosed herein can be used as dynamic sensing platforms that respond to changes in the surrounding environment or target analytes. By incorporating stimuli-responsive components (such as molecular switches or responsive polymers) into the scaffold design, researchers can engineer sensors that undergo conformational changes or signal modulation in the presence of specific analytes. This dynamic responsiveness enables real-time monitoring of molecular interactions and facilitates the development of biosensors for dynamic physiological processes or point-of-care diagnostics. Biocompatibility and stability: The self-assembling scaffolds disclosed herein are generally biocompatible and stable in biological environments, making them suitable for in vivo sensing applications and diagnostic tools. These scaffolds can be designed to withstand physiological conditions and minimize nonspecific interactions with biological matrices, thereby ensuring accurate and reliable detection of target molecules in complex biological samples. Furthermore, their stability allows for long-term monitoring and continuous sensing in vivo, enabling applications such as implantable biosensors or wearable diagnostic devices.

[0101] The self-assembling scaffolds disclosed herein can play a key role in advancing molecular robotics and computing by providing a programmable platform for building dynamic molecular systems and devices. For example: Bottom-up Assembly: The self-assembling scaffolds disclosed herein enable the bottom-up assembly of complex molecular structures and devices from simple building blocks. By engineering the molecular interactions and self-assembly pathways of the scaffold components, researchers can orchestrate the precise assembly of molecular machines, robots, and circuits with defined structures and functions. This bottom-up approach allows the creation of complex and sophisticated molecular systems with nanoscale precision, overcoming the limitations of top-down fabrication techniques. Dynamic Reconfigurability: The self-assembling scaffolds of the present disclosure offer dynamic reconfigurability, allowing for the reversible rearrangement of molecular components and structures in response to external stimuli or programmed inputs. By incorporating stimuli-responsive elements (such as switchable molecules or responsive polymers) into the scaffold design, researchers can engineer molecular systems that undergo programmable conformational changes or structural transformations on demand. This dynamic behavior enables molecular robotics and computing devices to be reprogrammed and adapted for a variety of different applications and tasks. Information processing and computing: The self-assembling scaffolds disclosed herein provide information processing and computing at the molecular level. By designing scaffolding architectures that support the organization and interactions of molecular components, researchers can use molecular logic gates, circuits, and networks to perform computational algorithms and logical operations. These molecular computing devices can perform complex tasks such as data storage, pattern recognition, and decision-making, opening up new possibilities for computation and information processing at the molecular level. Functional Integration: The self-assembling scaffolds disclosed herein facilitate the integration of functional components and modules into molecular robotics and computing devices. By incorporating diverse functionalities, such as sensors, actuators, and communication modules, onto the scaffold surface or within the scaffold structure, researchers can create multifunctional molecular systems capable of sensing, processing, and responding to environmental signals or external commands. This functional integration enhances the versatility and practicality of molecular robotics and computing devices in applications such as nanomedicine and materials science. Scalability and Reproducibility: The self-assembling scaffolds of the present disclosure offer scalability and reproducibility in the fabrication of molecular robotics and computing devices. Unlike conventional photolithography or micromanipulation techniques, which are limited by resolution and throughput, the self-assembly process is inherently parallel and scalable, allowing for the rapid and cost-effective generation of large numbers of identical molecular structures. This scalability and reproducibility are crucial for translating laboratory-developed prototypes into practical applications and commercial products.

[0102] Last but not least, the self-assembling scaffolds of the present disclosure can also be increasingly used in the field of bioprinting, where they offer unique advantages for the fabrication of complex 3D biological structures and tissues. Examples include: Support and structure: The self-assembling scaffolds disclosed herein provide a structural framework and support for bioprinted constructs. By self-assembling into ordered arrangements, these scaffolds provide a stable foundation for depositing bioink and building complex 3D structures layer by layer. This support is crucial for maintaining the integrity and shape fidelity of bioprinted tissues and organs, and preventing collapse or deformation during the printing process. Cell Encapsulation and Alignment: The self-assembling scaffolds disclosed herein facilitate the encapsulation and alignment of cells within bioprinted constructs. By incorporating cell-adhesive molecules or peptides onto the scaffold surface, researchers can promote cell adhesion and proliferation within the scaffold matrix, thereby enhancing cell viability and functionality. Furthermore, self-assembling scaffolds can align cells along specific orientations or patterns, mimicking the natural architecture of tissues and promoting cell-cell communication and organization. Biochemical Signaling and Microenvironment: The self-assembling scaffolds disclosed herein enable the incorporation of biochemical signaling and microenvironment into bioprinted constructs. By functionalizing scaffold components with bioactive molecules, growth factors, or extracellular matrix proteins, researchers can create biomimetic environments that support cell growth, differentiation, and tissue regeneration. These biofunctionalized scaffolds enhance the biological properties and functionality of bioprinted tissues, thereby promoting tissue maturation and integration within the host environment. Dynamic remodeling and degradation: The self-assembling scaffolds disclosed herein can exhibit dynamic remodeling and degradation properties. This allows bioprinted tissues to gradually remodel and integrate into host tissue. By incorporating degradable components or responsive polymers into scaffold designs, researchers can engineer scaffolds that undergo controlled degradation and remodeling in response to physiological signals or cellular activity. This dynamic behavior enables the scaffold material to be gradually replaced by the native extracellular matrix, thereby promoting tissue maturation and vascularization in bioprinted constructs. Multimaterial printing: The self-assembling scaffolds disclosed herein enable multimaterial bioprinting, in which multiple bioinks or scaffold materials are deposited simultaneously to create complex, heterogeneous tissues and organs. By designing scaffold structures that accommodate the deposition of multiple materials, researchers can create bioprinted constructs with spatially defined regions or gradients of biochemical and mechanical properties. This multimaterial printing capability enables the fabrication of functional tissues with heterogeneous cell populations, vascular networks, and specialized microenvironments, thereby advancing the development of complex organ models and regenerative therapies.

[0103] In another embodiment, the three-dimensional scaffolds of the present disclosure are contemplated for use as medicaments, such as for tissue and / or cell repair, tissue and / or cell engineering, drug delivery, wound treatment, bone reconstruction, construction of artificial organs, and combinations thereof.

[0104] Examples include:

[0105] The three-dimensional scaffold disclosed herein is suitable for repairing damaged cardiac tissue following a heart attack. The scaffold, composed of peptide sequences that promote cell adhesion and tissue regeneration, is injected into the infarcted area of ​​the heart. Once injected, the scaffold self-assembles into a 3D matrix that provides mechanical support and creates a favorable microenvironment for the recruitment and proliferation of cardiomyocytes. Over time, the scaffold degrades, allowing the newly formed tissue to integrate with the surrounding myocardium, ultimately restoring cardiac function.

[0106] The three-dimensional scaffold disclosed herein can be used in neural tissue engineering to promote axonal growth and regeneration in spinal cord injuries. The scaffold mimics the architecture of the extracellular matrix and provides physical guidance signals for axonal regeneration. Neuronal cells seeded onto the scaffold adhere and extend their neurites along the aligned nanofibers, thereby promoting axonal regeneration and functional recovery in injured spinal cord tissue.

[0107] The three-dimensional scaffolds disclosed herein can be used for targeted drug delivery to treat cancer. The scaffolds can encapsulate hydrophobic chemotherapeutic drugs within their cores. Functional groups on the micelle surface can be modified with targeting ligands that specifically recognize cancer cells. Following administration, the scaffolds accumulate at the tumor site through passive or active targeting mechanisms and release the encapsulated drugs, enabling localized and effective chemotherapy with reduced systemic toxicity.

[0108] The three-dimensional scaffold disclosed herein can be applied as an advanced wound dressing for diabetic foot ulcers. A scaffold containing bioactive peptide sequences that promote cell proliferation and angiogenesis is topically applied to the wound bed. Upon contact with wound exudate, the scaffold self-assembles to form a protective barrier that maintains a moist wound environment and accelerates tissue regeneration. The bioactive peptides in the scaffold stimulate the migration and proliferation of keratinocytes, fibroblasts, and endothelial cells, thereby promoting wound closure and angiogenesis. In this context, the scaffold can further incorporate antibiotics to inhibit bacterial growth and / or contain fibrinogen to promote integration of the scaffold with the natural coagulation process.

[0109] The three-dimensional scaffolds disclosed herein can be used for bone tissue engineering and reconstruction. The scaffolds, which contain biocompatible ceramic nanoparticles that self-assemble into a porous structure, provide a biomimetic environment for osteoblast seeding and bone formation. Osteogenic cells (such as mesenchymal stem cells) adhere to the scaffold surface and differentiate into osteoblasts, thereby depositing new bone matrix within the scaffold pores. Over time, the scaffold gradually degrades, leaving behind newly formed bone tissue that integrates with the surrounding bone, promoting bone regeneration and repair.

[0110] The three-dimensional scaffold of the present disclosure can be engineered to be used to build the functional unit of artificial organ or its part (such as kidney).The kidney organoids derived from induced pluripotent stem cell or primary nephrocyte are cultivated in the self-assembly scaffold of the extracellular matrix composition of simulation natural kidney tissue.This scaffold provides structural support and biochemical signal (for example, suitable growth factor, hormone, serum factor are arranged on this scaffold) for the growth and maturation of organoid.Along with the growth and differentiation of organoid, they reproduce the key feature of nephron, including filtration, reabsorption and secretory function, and finally form the functional unit that can be assembled into bioengineered kidney, for transplanting or in vitro drug testing.

[0111] In another embodiment, the present disclosure relates to a kit comprising a three-dimensional scaffold as disclosed below, further comprising a buffer, a package insert, an applicator, a dosing device, a mixing device, a manual, a device for inducing polymerization, a dye, and a hydrogel matrix, and combinations thereof.

[0112] Sequences of the present disclosure

[0113] and isoforms, variants and fragments thereof.

[0114] definition

[0115] Titin (SEQ ID NOs: 1-13), also known as connectin, is a giant protein found in vertebrate muscle cells. It is the largest known protein, with a molecular weight ranging from 3 to 4 megadaltons (MDa) and a length of up to 1 micron. Titin spans half the length of the sarcomere, the basic contractile unit of muscle, extending from the Z-disc to the M-line.

[0116] Functionally, titin plays several key roles in muscle physiology: Elasticity: Titin acts as a molecular spring, providing passive elasticity to muscle fibers and facilitating their ability to stretch and retract during contraction and relaxation. Its malleable structure enables it to withstand and transmit the forces generated during muscle contraction. Sarcomere assembly and stability: Titin anchors the myosin thick filaments to the Z-disc and helps maintain the structural integrity of the sarcomere. It interacts with other structural proteins such as actin and myosin. To stabilize the sarcomere structure and ensure the correct alignment of the contractile filaments. Regulation of muscle function: Titin plays a role in the regulation of muscle function by regulating the passive tension and stretching of muscle fibers in response to changes in muscle length. It facilitates the passive force generated during muscle lengthening and helps optimize muscle performance under a range of physiological conditions. Signaling: Titin is involved in signaling pathways involved in muscle growth, development, and adaptation to mechanical stress. It interacts with various proteins and signaling molecules, influencing processes such as muscle hypertrophy, mechanotransduction, and gene expression.

[0117] "PEVK" is an acronym that represents a region found in titin, a giant elastic protein found in muscle tissue. The PEVK region stands for proline (P), glutamic acid (E), valine (V), and lysine (K). These amino acids are part of a repeating sequence found in the I-band region of titin, which contributes to the protein's ductility and elasticity. The PEVK region is known to have a high content of proline and glutamic acid residues, which are thought to give the titin molecule its flexibility, allowing it to stretch and retract during muscle contraction and relaxation.

[0118] Actinin (SEQ ID NO: 14) is a large protein found in skeletal muscle fibers that, like titin, plays a crucial role in regulating the length of thin filaments in the sarcomere, thereby facilitating muscle contraction.

[0119] Obscurin (SEQ ID NOs: 15-19) is a giant sarcomeric protein present in skeletal and cardiac muscle. It interacts with various structural and signaling proteins (including titin) and participates in maintaining the structural integrity of muscle cells and regulating contractile function.

[0120] Dystrophin (SEQ ID NOs: 20-36) is a cytoskeletal protein primarily found in muscle cells. It plays a crucial role in connecting the cytoskeleton to the extracellular matrix and stabilizing the sarcolemma (cell membrane) during muscle contraction. Mutations in the dystrophin gene are associated with Duchenne muscular dystrophy and Becker muscular dystrophy.

[0121] As used below, the terms "protein isoform" and "protein variant" refer to different concepts related to proteins, especially variations in their structure, sequence, or function. The following is a brief explanation of each term:

[0122] "Protein isomers" or "isomers" refer to different spatial arrangements of atoms within a protein molecule while retaining the same chemical composition. Protein isomers can result from various types of isomerization processes, such as conformational changes, cis-trans isomerization of peptide bonds, or changes in the stereochemistry of amino acid residues. For example, in the case of conformers, these proteins can exist in multiple conformations or structural states due to the flexibility or dynamic mobility of the constituent amino acid residues of the protein. These conformers can have different shapes or folding patterns while having the same amino acid sequence. Protein isomers can also include stereoisomers, which have the same amino acid sequence but different spatial arrangements of their atoms. For example, cis-trans isomerization of proline residues in a protein chain can result in different spatial arrangements of the polypeptide backbone.

[0123] "Protein variants" hereinafter refer to different forms or versions of proteins produced due to genetic variation, post-translational modification, alternative splicing or other processes that cause changes in the amino acid sequence or structure of a protein. Protein variants can have changes in their amino acid sequence that cause amino acid substitutions, insertions, deletions or truncations, such as single nucleotide polymorphisms (SNPs). These variations can affect the structure, function, stability or interaction of proteins with other molecules. In one embodiment, a protein is considered to be a variant if it has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99.0%, at least 99.3%, at least 99.5%, at least 99.9% or at least 99.99% sequence identity to any of the sequences disclosed below. Additionally, protein variants can result from post-translational modifications (PTMs) such as phosphorylation, glycosylation, acetylation, or proteolytic cleavage, which can modify specific amino acid residues or alter the chemical properties and biological activity of the protein.

[0124] The term "substantially complementary nucleic acid sequences" refers to two nucleic acid (DNA or RNA) chains that have a sequence that can be paired with each other according to Watson-Crick (Watson-Crick) base pairing rules. In other words, the sequences are complementary in this way: adenine (A) in DNA is paired with thymine (T), adenine (A) in RNA is paired with uracil (U), and guanine (G) is paired with cytosine (C). The term "substantially complementary" means that the sequences may not be completely complementary over their entire length, but have enough complementary bases together to form a stable duplex structure. This can include sequences with accidental mispairing, protrusions, loops, or non-Watson-Crick base pairs, as long as overall complementarity allows stable hybridization and formation of a double-stranded structure under the hybridization conditions of selection. Substantially complementary nucleic acid sequences are commonly used in various molecular biology techniques, such as PCR (polymerase chain reaction), DNA sequencing, hybridization assays, and molecular cloning, where specific sequences need to anneal or hybridize to each other to achieve desired results. In one embodiment, "substantially complementary nucleic acid sequences" comprise at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% complementary nucleobases.

[0125] "Specific hybridization" of nucleic acids refers to the selective binding of two nucleic acid strands with complementary sequences to form a stable duplex structure. In specific hybridization, base pairing between nucleotides follows the Watson-Crick base pairing rules, where adenine (A) pairs with thymine (T) in DNA or uracil (U) in RNA, and guanine (G) pairs with cytosine (C).

[0126] The term "specific hybridization" refers to hybridization that occurs only between nucleic acid chains that have sequences sufficiently complementary to each other to allow precise and predictable interactions. Specific hybridization relies on the complementarity of nucleic acid sequences, where each base on one chain specifically pairs with its complementary base on the other chain. Specific hybridization allows one hybridization pair (e.g., A and A') to be distinguished from another hybridization pair (e.g., B and B') in solution under selected hybridization conditions and does not apply to "non-specific hybridization" (e.g., A and B).

[0127] In the context of the present disclosure, "barcode" refers to a short, unique sequence of nucleotides (DNA or RNA) used as a molecular identifier or tag. These barcodes are incorporated into nucleic acid molecules to distinguish and identify them in high-throughput experiments such as DNA sequencing, gene expression analysis, or multiplex assays. Barcode sequences are typically designed to be short enough to be easily read and identified, but long enough to provide sufficient diversity to distinguish between different samples or molecules. During library preparation or synthesis, barcodes can be added to nucleic acids using various molecular biology techniques such as PCR (polymerase chain reaction), ligation, or synthesis using modified nucleotides.

[0128] In the context of the present disclosure, "programmable" refers to the ability to design and manipulate nucleic acid sequences in a precise and predictable manner to perform specific tasks or functions. DNA molecules are programmable because the nucleotide sequences in the DNA chain can be precisely engineered to encode information or instructions for performing desired molecular processes. By designing complementary sequences and utilizing the Watson-Crick base pairing principle, researchers can create nucleic acid structures with predetermined shapes, properties, and functionality. In nucleic acid nanotechnology, programmable DNA sequences are used to construct nanostructures and nanodevices through self-assembly or molecular recognition processes. These nanostructures can be designed to have specific shapes, sizes, and surface properties, making them useful in applications such as drug delivery, biosensing, and molecular imaging. In DNA computing, programmable DNA sequences are used as information carriers and processing units to perform computing tasks. By encoding logic gates, algorithms, or computational instructions into DNA sequences, researchers can use DNA molecules to perform parallel computing and solve complex computational problems.

[0129] In the context of this disclosure, "PDI" stands for "polydispersity index." The polydispersity index is a measure of the molecular weight distribution in a polymer sample. It provides information about the spread or variability of molecular weight within a sample. The polydispersity index is calculated by dividing the weight average molecular weight (Mw) by the number average molecular weight (Mn). Mathematically, it can be expressed as: in: M w is the weight average molecular weight, calculated by adding the product of the molecular weight of each polymer chain and its corresponding weight fraction in the sample; and M n is the number average molecular weight, calculated by adding the product of the molecular weight of each polymer chain and its corresponding number fraction in the sample.

[0130] A polydispersity index of 1 indicates a monodisperse polymer sample in which all polymer chains have the same molecular weight. A higher polydispersity index indicates a broader molecular weight distribution and greater variability between polymer chains in the sample.

[0131] The polydispersity index is an important parameter in polymer characterization because it affects the physical properties and performance of polymeric materials. Polymers with lower polydispersities generally exhibit more uniform properties and behavior, while polymers with higher polydispersities can have more variable properties and performance due to the wider molecular weight range.

[0132] In one embodiment, the three-dimensional scaffold may have a PDI of at least 1.0, at least 1.1, at least 1.2, at least 1.5, at least 2.0, at least 3.0, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, or at least 10.0.

[0133] To test the "tensile strength" of proteins, a technique called "mechanical stretching" or "tensile testing" can be used. This method involves applying a controlled mechanical force to a protein sample and measuring its response to deformation. While this method is typically used to test the mechanical properties of materials, such as polymers and metals, it can also be applied to proteins.

[0134] The main steps in measuring the tensile strength of a protein are: Sample preparation: Protein samples need to be prepared into a form suitable for tensile testing. This can include isolating the protein of interest and shaping it into a consistent and homogeneous structure such as a fiber, film, or hydrogel. Samples should be free from defects or impurities that may affect the test results. Mounting the sample: Mount the prepared protein sample on a test device equipped with a clamp or clip. These clamps or clips hold the sample securely. Care should be taken to ensure that the sample is properly aligned and The clamp applies evenly distributed force to prevent premature failure or deformation. Applying force: A controlled mechanical force is applied to the protein sample using a testing device. This force is gradually increased at a constant rate while the deformation of the sample is monitored. The applied force can be directly measured using a load cell integrated into the testing machine. Deformation measurement: During tensile testing, the deformation of a protein sample is typically measured using displacement transducers or extensometers. These devices track the change in length or dimension of a sample as it undergoes stretching or elongation. Analysis of stress-strain curves: Data obtained from tensile testing (including applied force and deformation measurements) are used to generate stress-strain curves for protein samples. Stress is calculated as the applied force per unit area, while strain is the ratio of the change in length of the sample to its initial length. The tensile strength of a protein, which represents its ability to resist deformation under tension, can be determined from the maximum stress observed on the stress-strain curve.

[0135] Measuring the ductility of a protein involves quantifying its ability to deform or elongate in response to an applied force.Ductility is typically assessed experimentally using techniques such as atomic force microscopy (AFM), optical tweezers, or mechanical stretching assays.

[0136] AFM is a powerful technique that allows for the direct manipulation and measurement of the mechanical properties of individual protein molecules. In an AFM-based stretching experiment, the protein of interest is typically immobilized on a surface, and a cantilever with a pointed tip is brought into contact with the protein. As the cantilever moves, the protein molecule is pulled or stretched by it, and the resulting force-displacement curve is recorded. From this curve, parameters such as the protein's ductility, stiffness, and unfolding force can be determined.

[0137] Measuring the toughness of a protein involves assessing its ability to absorb energy and deform without breaking when subjected to mechanical stress. Toughness is a key mechanical property that reflects the ability of a protein to resist breaking or failing under load. AFM-based techniques can be used to measure the toughness of proteins by subjecting individual protein molecules to mechanical deformation and monitoring their responses. In an AFM force spectroscopy experiment, a protein molecule is fixed to a surface and a cantilever with a tip is brought into contact with the protein. The cantilever is then moved to apply a controlled force to the protein, causing it to deform. The toughness of the protein can be determined from the force-displacement curves obtained during the stretching or unfolding process. Proteins with higher toughness will require more energy to deform and will exhibit higher resistance to breaking.

[0138] Further methods for testing the physical properties of proteins, such as tensile strength, ductility, and toughness, can be found in Bowen et al., “Microbial production of megadalton titin yields fibers with advantageous mechanical properties,” Nature communications, https: / / doi.org / 10.1038 / s41467-021-25360-6.

[0139] Example

[0140] Example 1 - Production of Stent Element (101)

[0141] 1.1 Strains and growth conditions

[0142] All plasmid cloning and protein production were performed using Escherichia coli NEB 10-β (NEB10β). For all clones, E. coli strains were cultured at 37°C in Terrific Broth (TB) containing 24 g / L yeast extract, 20 g / L tryptone, 0.4% v / v glycerol, 17 mM KH2PO4, and 72 mM K2HPO4 with appropriate antibiotics (50 μg / mL kanamycin). M9 glucose medium with tryptone supplement (2% w / v glucose, 1× M9 salts, 75 mM MOPS pH 7.4, 12 g / L tryptone, 5 mM sodium citrate, 2 mM MgSO4 7H2O, 100 μM FeSO4 7H2O, 100 μM CaCl22H2O, 3 μM thiamine, 1× micronutrients (40 μM ZnSO4 7H2O, 20 μM CuSO4 5H2O, 10 μM MnCl2 4H2O, 4 μM H3BO3, 0.4 μM (NH4)6Mo7O) 24 4H2O and 0.3 μM CoCl2 6H2O) were used for protein production in bioreactors.

[0143] 1.2 Chemicals and reagents

[0144] Unless otherwise stated, all chemicals and reagents were obtained from Millipore and Sigma. Plasmid purification and gel extraction kits were purchased from iNtRON Biotechnology. FastDigest restriction enzymes and T4 DNA ligase were purchased from ThermoFisher Scientific and used for all digestions and ligations according to the manufacturer's protocols.

[0145] 1.3 Construction and expression optimization of titin-based peptide components (102) for scaffold elements (101)

[0146] The coding sequence of titin SEQ ID NO: 1 was inserted between the KpnI and Kpn2I restriction sites of the modified BglBricks63 vector containing gp41-1C and gp41-1NSIs, under the control of the PBAD promoter or the PLAcO1 promoter, to obtain an expression plasmid.

[0147] 1.4 Bioproduction in shake flasks

[0148] Incubate 50 mL of overnight seed culture in TB medium with a single colony carrying the desired construct. This seed culture is then used to inoculate a 500 mL TB culture in a 2-L Erlenmeyer flask to an initial OD600 of 0.08. The culture is incubated on a reciprocating shaker at 350 rpm and 37°C until the OD600 reaches 3.0, at which point the appropriate inducer is added. Cultivation is then continued at 37°C for 20 hours.

[0149] 1.5 Bioproduction in fed-batch bioreactors

[0150] Finally, titin-based peptide components (102) were produced in 2 L fed-batch bioreactors (Bioflo 120, Eppendorf). Transformants containing the plasmid were cultured overnight in 50 mL of TB medium on an orbital shaker at 37°C. The overnight culture was then used to inoculate an autoclaved 2 L Bioflo 120 heat-blanketed bioreactor containing 1.5 L of M9 glucose medium with tryptone supplement (see above). Antifoam 204 was added as needed to minimize foaming (approximately 0.01% v / v). Agitation and airflow were regulated to maintain a dissolved oxygen (DO) of approximately 70%.

[0151] After the initial 0.5% w / v glucose (judged by ΔDO) was consumed, sterile substrate feeding (20% w / v glucose, 48 g / L tryptone and 10 g / L MgSO 4 7H 2 O) was started to maintain a linear growth rate. At OD600=70, the reactor was induced by adding 1 mM IPTG and the incubation temperature was reduced to 30° C.

[0152] Cultures were harvested six hours after induction.

[0153] 1.6 Protein purification

[0154] The titin-based peptide fraction (102) was purified by dissolving the cell pellet in aqueous lysis buffer (50 mM Tris, 50 mM NaCl, 1 mM PMSF, and 300 μg / mL lysozyme). After stirring at 4°C for 30 min, 5 mM MgCl2 and 5 μg / mL DNase I were added and the mixture was sonicated while stirring on ice for 10 min (5 s on, 10 s off). After sonication, NaCl and imidazole were added to final concentrations of 300 mM and 10 mM, respectively. The mixture was centrifuged at 25,000 × g for 30 min at 4°C, followed by centrifugation at 75,000 × g for 30 min at 4°C. The clarified supernatant was then filtered and applied to a series of six HisTrap HP 5 mL columns at 2 mL / min. The loaded column was washed with 2 column volumes of wash buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole), then washed with 2 column volumes of wash buffer containing 50 mM imidazole, and finally eluted with wash buffer containing 300 mM imidazole.

[0155] After purification by affinity chromatography, the protein (ie, titin-based peptide fraction (102)) was extensively dialyzed against 5 mM ammonium bicarbonate at 4°C using 10 kDa MWCO snakeskin dialysis tubing (Thermo Fisher Scientific).

[0156] 1.7 Adding two nucleic acid components (103, 104)

[0157] Two nucleic acid components of SEQ ID NO: 37 and 38 were added to either end of the titin-based peptide component (102) by strain-promoted alkyne-azide cycloaddition (SPAAC).

[0158] exist Figure 11 The final stent element (101) is schematically depicted in FIG.

[0159] Example 2 - Formation of a three-dimensional scaffold

[0160] About 0.5% (w / v) of the scaffold elements (101) were added to aqueous sodium phosphate buffer (pH 7.5) at 20° C. The solution was stirred moderately and then stored for 6 hours to allow self-assembly until the three-dimensional scaffold was formed.

[0161] The three-dimensional scaffold can be formed into a certain form by using a suitable container for self-assembly, or by cutting the three-dimensional scaffold after forming, for example, using a laser cutter.

Claims

1. A three-dimensional scaffold (100), comprising: A plurality of scaffold elements (101), each scaffold element comprising at least one amino acid peptide component (102) and at least two nucleic acid components (103, 104), wherein the peptide component is composed of a stretch of amino acids, wherein the at least two nucleic acid components (103, 104) of the plurality of scaffold elements (101) are configured to mediate self-assembly of the scaffold (100).

2. A three-dimensional scaffold according to claim 1, wherein the at least two nucleic acid components (103, 104) include a first nucleic acid component and a second nucleic acid component, the second nucleic acid component is different from the first nucleic acid component, and the first nucleic acid component and the second nucleic acid component are preferably constructed to hybridize with different targets.

3. The three-dimensional scaffold according to any one of the preceding claims, wherein the at least one peptide component (102) does not comprise PNA.

4. The three-dimensional scaffold according to any one of the preceding claims, wherein the at least one peptide component (102) consists of a stretch of amino acids connected by peptide bonds.

5. The three-dimensional scaffold according to any of the preceding claims, wherein at least two nucleic acid components (103, 104) are located at predetermined positions of the scaffold element (101).

6. The three-dimensional scaffold according to any of the preceding claims, wherein each of the at least two nucleic acid components (103, 104) comprises 9 to 25 nucleobases.

7. A three-dimensional scaffold according to any of the preceding claims, wherein each of the at least two nucleic acid components (103, 104) is encoded to specifically hybridize with at least one of the nucleic acid components of another scaffold element (101a, 101b), thereby mediating self-assembly of the scaffold (100).

8. The three-dimensional scaffold according to any one of the preceding claims, wherein the at least one peptide component (102) is characterized by at least one of the following features at a molecular weight (MW) of 2.5 MDa: · Longitudinal tensile strength of at least 350 MPa, A tensile modulus of at least 3.5 GPa, At least 45% ductility, At least 120Mj / m 3 Resilience, having a length of at least 0.5 μm, Contains at least 3000 amino acids.

9. The three-dimensional scaffold according to any one of the preceding claims, wherein the at least one peptide component (102) comprises at least 100 Ig-like domains and / or at least 10 Proline-Glutamic Acid-Valine-Lysine (PEVK) motifs.

10. The three-dimensional scaffold according to any one of the preceding claims, wherein the at least one peptide component (102) comprises a peptide selected from SEQ ID NO: 1 to 36, and variants having at least 90% sequence identity, and isoforms thereof.

11. The three-dimensional scaffold according to any one of the preceding claims, further comprising at least one nucleic acid-based binding region (105) and / or at least one peptide-based binding region (106).

12. A method for producing a three-dimensional scaffold, comprising the steps of: a. providing a plurality of scaffold elements (101), said scaffold element (101) comprising at least one peptide component (102) and at least two nucleic acid components (103, 104), b. Among them, Optionally, at least one of the scaffold elements (101) further comprises a binding element selected from a nucleic acid-based binding region (105) and / or a peptide-based binding region (106), c. placing the scaffold elements in a solution, whereby self-assembly of the scaffold elements (101) occurs, and d. Obtain a three-dimensional scaffold.

13. Use of the three-dimensional scaffold according to any one of claims 1 to 11, wherein the use is selected from: use in biological imaging and labeling, use in templates for material synthesis, use in molecular sensing, use in diagnostic tools, use in molecular robotics and computing, use in synthetic biology, use in bottom-up nanomanufacturing, use in nanoscale devices, use in bioprocessing and use in bioprinting and combinations thereof.

14. The three-dimensional scaffold according to any one of claims 1 to 11 for use as a medicament, such as for the treatment of tissue and / or cell repair, tissue and / or cell engineering, drug delivery, trauma, bone reconstruction, construction of artificial organs, and combinations thereof.

15. A kit comprising the three-dimensional scaffold according to any one of claims 1 to 11, further comprising at least one item selected from the group consisting of a buffer, a package insert, an applicator, a dosing device, a mixing device, a manual, a device for inducing polymerization, a dye, and a hydrogel matrix, and combinations thereof.

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