Conformational switching nanostructures

Abstract

The present invention relates to conditionally switchable nanostructures and systems comprising same. In particular, the present invention relates to a method of relating the identification of at least one molecule to the conformational rearrangement of the nanostructure. The present invention is further based on the finding that coupling molecules, such as antibodies or antigen-binding fragments, can stabilize the subunit interactions of the nanostructure in otherwise unfavorable conditions. Furthermore, the present invention also relates to systems comprising the nanostructures of the invention as well as methods of utilizing the nanostructures or systems of the invention. Finally, substances and / or compositions comprising at least one or a plurality of nanostructures for use as a medicament or for use in diagnostic methods are also provided.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to conditionally switchable nanostructures and systems comprising same. In particular, the present invention relates to a method of relating the identification of at least one molecule to the conformational rearrangement of the nanostructure. The present invention is further based on the finding that coupling molecules, such as antibodies or antigen-binding fragments, can stabilize the subunit interactions of the nanostructure in otherwise unfavorable conditions. Furthermore, the present invention also relates to systems comprising the nanostructures of the invention as well as methods of utilizing the nanostructures or systems of the invention. Finally, substances and / or compositions comprising at least one or a plurality of nanostructures for use as a medicament or for use in diagnostic methods are also provided. [Background technology]

[0002] explanation Molecular recognition is a fundamental feature of biological systems and is required, for example, for the recognition of T cells by the adaptive immune system or the transmission of molecular signals across cell membranes. An important advance in modern medicine is the development of therapeutic agents that demonstrate similar molecular recognition properties, such as therapeutic antibodies that target specific cell surface receptors. Recent advances in the field of DNA nanotechnology have made it a promising candidate for the development of "intelligent" nanoscale devices that exhibit molecular recognition properties similar to those observed in naturally occurring molecules. To develop such intelligent nanostructures, molecular recognition must be coupled to signaling mechanisms to confer conformational rearrangements that typically, but not exclusively, result in the release of cargo from a drug carrier or the activation of a therapeutic agent.

[0003] In recent years, new methods for producing nanostructures have been developed. These include the method for producing nucleic acid nanostructures disclosed in US Pat. No. 7,842,793 B3. The disclosure relates to a technique called "DNA origami", which is a method for producing nanoscale devices, systems, and enzyme factories based on nucleic acid nanostructures that can be designed to have a predetermined structure. DNA origami uses DNA molecules for the production of three-dimensional structures on the nanometer scale. Chemical modifications or single-stranded DNA overhangs can be placed on the origami structures to design and build the desired structure of the nucleic acid nanostructure, and the DNA origami structures can be linked to other molecules. The DNA origami structures can therefore be used to regulate cell signaling mechanisms.

[0004] Recently, DNA origami technology has been used to manufacture various nanoscale devices. As an example, WO 2012 / 061719A2 discloses a DNA origami device that is useful in the targeted delivery of biologically active entities to specific cell populations. This device can enable the targeted delivery of biologically active entities, while relying on specific interacting molecules, particularly aptamers, that can bind to two different binding targets.

[0005] US Patent No. 9,863,930 B2 discloses various molecular barcoded bistable switches that can be used to detect different analytes. In particular, the molecular barcoded bistable switches can be fabricated using DNA origami.

[0006] However, the integration of modules that control nanoparticle rearrangements induced by molecular signals remains a largely unmet challenge. To date, most of the few developed DNA origami nanoparticles that exhibit any molecular rearrangements rely on structure-switching aptamers. Typically, these aptamers lock the structure into a metastable closed configuration. Binding of an antigen to the aptamer closes induces an equilibrium transition to the open state. An inherent limitation of the use of structure-switching aptamers is this complex balance between open and closed state stability, which requires extensive tuning for different structures and aptamer types. Moreover, only a limited number of aptamers are known for an even smaller number of antigens, which typically bind with moderate affinity or require exogenous metal ions for proper function. Finally, aptamers exhibit a large structural heterogeneity, which makes modular design inherently challenging.

[0007] In view of the above, there is a continuing need in the art to develop nanostructures that allow molecular recognition to be coupled to signal transduction mechanisms. There is also an urgent need for the development of nanostructures that can recognize a large number of molecules and reliably change their conformation upon recognition. Furthermore, there is also an urgent need for nanostructures that bind to target molecules with high affinity. It is an object of the present invention to overcome or at least mitigate the shortcomings and disadvantages of the prior art. That is, it is an object of the present invention to provide a novel conformation-switching nanostructure. Summary of the Invention

[0008] Brief description of the invention Generally, and by way of brief description, the main aspects of the present invention can be described as follows:

[0009] In a first aspect, the present invention relates to a nanostructure comprising at least two coupling sites.

[0010] In a second aspect, the present invention relates to a system comprising a nanostructure.

[0011] In a third aspect, the present invention relates to a method for relating the identification of at least one molecule to the conformational reconfiguration of a nanostructure.

[0012] In a fourth aspect, the present invention relates to a substance comprising at least one or a plurality of nanostructures for use as a medicament or for use in a diagnostic method.

[0013] In a fifth aspect, the present invention relates to a composition comprising at least one or a plurality of nanostructures for use as a medicament or for use in a diagnostic method. [Brief description of the drawings]

[0014] Brief Description of Figures and Sequences The figure shows: [Figure 1]Figure 1 shows IgG-mediated stabilization and antigen-induced disassembly of icosahedral DNA origami shells. Strategically attached antigens on DNA origami subunits ("entities", shown as triangular shapes) allow bivalent antibodies ("coupling molecules") to act as molecular bridges between different DNA origami subunits ("entities", shown as triangular shapes) to stabilize subunit interactions in otherwise unfavorable conditions when bound to antigen fragments. Shape-complementary protrusions and recesses govern the self-assembly of icosahedral shells from 20 identical copies of multilayered DNA origami subunits. Self-assembly of icosahedral shells requires the addition of Mg2+ ions to stabilize blunt-ended stacking contacts between the shape-complementary features. Antibody-specific antigens are installed in symmetrical positions on the DNA origami subunit ("triangle") monomer surface. Shell assembly results in the placement of antigens on the opposite sides of each triangle-triangle interface. An antigen-specific antibody ("coupling molecule") bivalently binds to the antigen attached to the shell, stabilizing the triangle-triangle interaction. Subsequent dilution to Mg2+ ion concentrations that would otherwise destabilize the icosahedral shell leaves it in a metastable "spring-loaded" state. Controlled disassembly is triggered by the addition of a co-antigen ("competitor antigen") that displaces the stabilizing antibody from the shell, thereby inducing a conformational rearrangement of the DNA origami structure. Legend: Mg2+ = magnesium ion; 1 triangle = 1 DNA origami subunit ("entity"); "coupling site" = antigen fragment (preferably attached onto the DNA origami subunit via a staple strand); antibody = "coupling molecule". [Diagram 2]FIG. 2 shows the structural characterization and evaluation of Mg2+-dependent IgG-mediated shell stabilization. (a) Top: Exemplary Cryo-EM micrograph of an icosahedral shell in free-standing ice with anti-digoxigenin antibodies bridging the triangle-triangle interface at 25 mM MgCl2. Bottom: 2D class averages from different orientations. (b) Cryo-EM reconstruction of an assembled icosahedral shell with two anti-digoxigenin antibodies bridging each triangle-triangle interface. (c) (Left) Zoom-in on the triangle-triangle interface bridged by two anti-digoxigenin antibodies depicted in the cylinder model (PDB:1IGT) with manually placed IgG antibodies and (right) Cryo-EM reconstruction. Notably, both Fab fragments, as well as parts of the Fc region of the antibodies, are reconstructed. (d) Schematic of antibody-mediated stabilization at reduced Mg2+ concentrations. (e) Laser-scanned fluorescence images of 0.75% agarose gels in 0.5x TBE buffer supplemented with 8, 12, 16 and 20 mM MgCl2. Assembled shells were incubated with or without anti-digoxigenin antibody at 25 mM MgCl2, subsequently diluted to each MgCl2 concentration and electrophoresed on the corresponding gel. Legend: Monomer = single triangular monomer, Dimer = dimer formed by two triangular subunits, Shell = fully assembled icosahedral shell. (f) Negative stain TEM images of shells incubated without (left) or with (right) anti-digoxigenin antibody and diluted to 12 mM MgCl2. (g) Normalized FRET ratios of shells incubated without and with anti-digoxigenin antibody and subsequently diluted to various final MgCl2 concentrations. Each side of the triangular subunit is functionalized with Cy3 and Cy5 fluorophores at symmetrical positions, which results in energy transfer only when the shell is assembled. The initial decrease in the FRET ratio at MgCl2 concentrations between 22 and 14 mM is caused by the swelling of the shell due to increasing electrostatic repulsion. [Diagram 3]FIG. 3 shows antigen-induced disassembly of icosahedral shells. (a) Addition of soluble digoxigenin ligand that competes for binding with shell-stabilizing anti-digoxigenin antibody displaces the antibody from the spring-loaded shell, which in turn induces shell disassembly at destabilizing Mg2+ ion concentrations. Laser-scanned fluorescent images of 0.75% agarose gel in 0.5×TBE buffer supplemented with 12 mM MgCl2. Assembled shells with anti-digoxigenin antibody were incubated with increasing concentrations of digoxigenin. Anti-digoxigenin-stabilized shells disassemble with increasing concentrations of soluble digoxigenin antigen. Negative stain TEM images of anti-digoxigenin-stabilized shells in the absence (left) and presence (right) of 25 μM soluble digoxigenin. (b) As in (a) except with anti-dinitrophenol-stabilized shells and addition of soluble 2,4-dinitrophenol ligand. (c) Normalized FRET ratios of shells stabilized by anti-digoxigenin with increasing concentrations of soluble digoxigenin ligand. (d) Normalized FRET ratios of shells stabilized by anti-dinitrophenol with increasing concentrations of soluble 2,4-dinitrophenol. The dashed lines represent the nonlinear least-squares optimization of the Hill equation to the experimental data. (e) Left: Shell disassembly gated by AND gate logic is achieved by placing both digoxigenin and 2,4-dinitrophenol ligands and their respective antibodies on the triangle-triangle interface. AND-gated shell disassembly therefore requires the presence of both soluble digoxigenin and 2,4-dinitrophenol. Right: Laser-scanned fluorescence images of 0.75% agarose gel in 0.5× TBE buffer supplemented with 12 mM MgCl2. Assembled AND gate shells with anti-digoxigenin and anti-dinitrophenol antibodies were incubated with increasing concentrations of top: digoxigenin, middle: 2,4-dinitrophenol and bottom: digoxigenin and 2,4-dinitrophenol.Negative stain TEM images of shells with anti-digoxigenin and anti-dinitrophenol incubated with top: 25 μM digoxigenin, middle: 5 mM 2,4-dinitrophenol and bottom: 25 μM digoxigenin and 5 mM 2,4-dinitrophenol. (f) Normalized FRET ratio of AND-gated shells stabilized by a combination of anti-digoxigenin and anti-dinitrophenol and incubated with a combination of soluble digoxigenin (25 μM) and 2,4-dinitrophenol (5 mM). [Figure 4] Figure 4 shows antigen-induced release of viral payload. (a) Scheme showing (from left to right) functionalization of a subset of triangles with Hepatitis B virus (HBV) core capsid, where binding of HBV particles is mediated by specific antibodies at the inward surface of the triangular shell; assembly of the complete shell with HBV core capsid in the lumen; stapling by anti-Dig IgG antibodies and spring loading by lowering ionic strength; antigen-induced shell rupture and release of viral payload by adding soluble digoxigenin antigen. Not shown is the intermediate step of detaching the HBV-binding antibodies located in the interior of the shell from the shell, leaving the HBV particles free floating in the shell. (b, c, d) Negative stain TEM images of shells assembled with 40 mM MgCl2 in the presence of viral payload, anti-Dig stapled shells loaded with viral payload under spring-loaded conditions (12 mM MgCl2), and shells disassembled by adding soluble digoxigenin antigen leading to the release of viral payload, respectively. Scale bars in (b, c) are 50 nm and in (d) are 100 nm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description of the Invention In the following, the elements of the present invention are described. These elements are listed with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the embodiments explicitly described. This description should be understood to support and encompass embodiments that combine two or more of the explicitly described embodiments or combine one or more of the explicitly described embodiments with any number of disclosed and / or preferred elements. Moreover, unless the context indicates otherwise, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application.

[0016] In a first aspect, the present invention relates to a nanostructure comprising at least two coupling sites, said at least two coupling sites being capable of binding to at least one coupling molecule (i.e. to one coupling molecule or to multiple coupling molecules), said at least two coupling sites being configured as a coupling site set.

[0017] In a preferred embodiment, a coupling site set is configured to bind to one coupling molecule. In general, it should be understood that a nanostructure comprising at least two coupling sites should not be interpreted as meaning that the nanostructure comprises only these two coupling sites and one coupling site set is configured to bind to only one coupling molecule. Instead, a nanostructure can also have more than two coupling sites and one coupling site set can be configured to bind to more than one coupling molecule.

[0018] In another preferred embodiment, the nanostructure comprises at least one first entity and at least one second entity, wherein each of the at least one first entity and the at least one second entity comprises at least one coupling site of at least one coupling site set, and preferably, the at least one coupling site of the at least one first entity and the at least one coupling site of the at least one second entity are located on symmetrical positions of the at least one first entity and the at least one second entity.

[0019] In a preferred embodiment, the coupling site set may only comprise identical coupling sites, i.e., coupling sites configured to couple to the same coupling molecule. In other embodiments, the coupling site set may comprise at least two separate coupling sites configured to couple to the coupling molecule by different interactions. The coupling site set may be formed by two coupling sites. However, in other embodiments, the coupling site set may also be formed by multiple coupling sites.

[0020] This invention generally relates to nanostructures. In the present example, the nanostructure may be a DNA origami structure, i.e., a DNA origami device, as exemplarily disclosed in U.S. Pat. No. 7,842,793 B2. However, it should be understood that the present technology is not limited thereto, and that this is merely exemplary and that other nanostructures may also be utilized to practice the present invention. However, the term "nanostructure" as used herein preferably refers to a DNA origami structure that is composed of multiple DNA origami subunits (entities). In some embodiments, the interior of the nanostructure of the present invention may be hollow and may contain molecules, such as therapeutic molecules and / or cargo molecules.

[0021] The term "entity" is intended to refer to nanostructures and / or DNA origami subunits.

[0022] The term "coupling site" is intended to refer to a site on a subunit ("entity") of a nanostructure to which a coupling molecule binds. Preferably, the coupling site is an antigen, an antigen fragment, or an antigen mimic. Alternatively, the coupling site is a biotin molecule, a streptavidin molecule, or any other molecule that specifically binds to a particular different molecule.

[0023] As used herein, a "coupling site set" refers to a coupling site set consisting of at least two, preferably two, coupling sites. Preferably, the coupling molecules are configured to couple one subunit (e.g., a "first entity") of the nanostructure to a second subunit (e.g., a "second entity") when bound to one coupling site set (e.g., by binding of an antibody to two antigen-binding constructs on different DNA origami subunits).

[0024] The term "at least one" according to the present invention is intended to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any other number. For example, the term "at least one" first entity is intended to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any other number of first entities. Similarly, the term "at least one" second entity is intended to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any other number of second entities.

[0025] In yet another embodiment, the at least one coupling molecule is configured to couple at least one first entity of the nanostructure to at least one second entity of the nanostructure when the at least one coupling molecule is bound to the at least one coupling site set.

[0026] The term "coupling molecule" refers to a molecule that binds to a coupling site. Preferably, the coupling molecule is an antigen-binding construct, such as an antibody. Alternatively, the coupling site is a streptavidin molecule or any other molecule that specifically binds to a different molecule. In the main assay of this invention, the coupling molecule is artificially added to the assay. The coupling molecule can reversibly bind to the coupling site, i.e., the coupling molecule can repeatedly reversibly bind to and separate from the coupling site. Thus, the coupling molecule can repeatedly couple and decouple to the coupling site.

[0027] Also preferred are nanostructures having at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any number of at least one first entity and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any number of at least one first entity and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any number of at least one second ... In another aspect, the present invention is intended to include a plurality of at least one first entity and a plurality of at least one second entity, such as 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any number of at least one second entity.

[0028] A further embodiment relates to the nanostructure of this invention, wherein the nanostructure comprises a plurality of coupling moiety sets, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any number of coupling moiety sets, preferably each coupling moiety set consisting of two coupling moieties.

[0029] Yet another embodiment relates to a nanostructure of the invention, wherein a coupling molecule bound to a first coupling site set of the plurality of coupling sites is the same coupling molecule bound to a second coupling site set of the plurality of coupling sites, or a coupling molecule bound to the first coupling site set of the plurality of coupling sites is a different coupling molecule from a coupling molecule bound to the second coupling site set of the plurality of coupling sites.

[0030] Nanostructures can be configured to assume different configurations, including at least one first configuration (A) and at least one second configuration (B). In some embodiments, these configurations can also be referred to as "open" and "closed" configurations. Depending on the configuration the nanostructure assumes, the nanostructure can vary the accessibility of different portions of the nanostructure. For example, the interior of the nanostructure can be more accessible in the second configuration (B) than in the first configuration (A).

[0031] In a preferred embodiment, the nanostructures are configured to have at least one first configuration (A), and at least one second configuration (B), optionally at least one third configuration (C), at least one fourth configuration (D), or any other number of configurations, i.e., any nanostructure of the embodiments of this invention can have different configurations (e.g., configurations (A), (B), (C), (D), etc.).

[0032] Depending on whether the coupling sites are linked to coupling molecules, the likelihood of the nanostructure to adopt different configurations may change. As an example, when all (or some) of the coupling sites are coupled by coupling molecules, the nanostructure may be more likely to adopt, for example, the first configuration (A) than when none of the coupling sites are coupled by coupling molecules. That is, depending on the coupling state of the coupling sites, the accessibility of the interior of the nanostructure may change. Thus, the coupling state of the coupling sites may "open" and / or "close" the nanostructure. It will be understood that this may also include the release of molecules, such as cargo molecules and / or therapeutic molecules, that may be present within the interior of the nanostructure.

[0033] The nanostructures may be configured to adopt a first configuration (A) with a higher probability than a second configuration (B) when at least one coupling molecule is bound to at least one respective coupling site set. Particularly preferred embodiments therefore relate to nanostructures of the invention, wherein the nanostructures are configured to adopt at least one first configuration (A) when at least one coupling molecule is bound to at least one coupling site set, and / or the nanostructures are configured to adopt at least one second configuration (B) when at least one coupling molecule is not bound to at least one coupling site set.

[0034] However, the nanostructure may also adopt both the first configuration (A) and the second configuration (B) when no coupling molecule is bound to the respective coupling site set. More specifically, there may be an equilibrium between the first configuration (A) and the second configuration (B). In this equilibrium, the first configuration (A) may be "preferred", i.e., the nanostructure may adopt this first configuration (A) more likely than the second configuration (B), or vice versa. Furthermore, the nanostructure may be configured to adopt at least one or more equilibrium states between the first configuration (A) and the second configuration (B). For example, the nanostructure may be configured to adopt a first and a second equilibrium state between the first configuration (A) and the second configuration (B), where the probability of the nanostructure adopting the second configuration (B) may be different (e.g., higher) in the second equilibrium state than in the first equilibrium state.

[0035] It will be appreciated that the above is merely an example and that the first and second equilibrium states may have different probabilities of assuming the first configuration (A) and the second configuration (B). In general, the second configuration (B) is more likely to be assumed in the second equilibrium state than in the first equilibrium state.

[0036] In another embodiment, the nanostructures are configured to adopt at least one first configuration (A) when little or no at least one competitor molecule is present, and / or the nanostructures are configured to adopt at least one second configuration (B) when a greater amount of the at least one competitor molecule is present.

[0037] That is, when a nanostructure is in an environment where there is little or no at least one competing molecule, such nanostructure is more likely to have configuration (A). Conversely, when a nanostructure is in an environment where there is a greater amount of at least one competing molecule, such nanostructure is more likely to have configuration (B). In general, a nanostructure may have a first configuration (A) when each coupling site, or a subset of a set of coupling sites, is coupled to its respective coupling molecule. At least one or a number of competing molecules may, however, induce and / or trigger the release of at least one coupling molecule. Thus, when a nanostructure is in an environment where there is a greater amount of at least one competing molecule, at least one coupling molecule is more likely to be released from the nanostructure. In such an event, the nanostructure may, for example, be rearranged from configuration (A) to configuration (B).

[0038] The term "competitor molecule" as used herein refers to a molecule that is preferably an antigen, an antigen fragment, or an antigen mimic. Alternatively, the competitor molecule can be a biotin molecule, a streptavidin molecule, or any other molecule that specifically binds to a specific different molecule. The competitor molecule competes with the coupling site for binding to at least one coupling molecule. The competitor molecule can be present in a sample, cell, organism, etc., such as a body fluid sample. It is one object of this invention to detect the competitor molecule.

[0039] In general, it will be understood that nanostructures can also adopt configuration (A) (e.g., a closed configuration) and / or configuration (B) (e.g., an open configuration) when the coupling moieties are uncoupled, partially coupled, or fully coupled to their respective coupling molecules.

[0040] Yet another preferred embodiment relates to the nanostructure of the present invention, wherein the nanostructure is configured to adopt at least one first configuration (A) in a first solution, which preferably contains a higher concentration of magnesium ions compared to a second solution, and / or the nanostructure is configured to adopt at least one second configuration (B) in a second solution, which preferably contains a lower concentration of magnesium ions compared to the first solution.

[0041] However, it should be understood that the conditions of the first solution and the second solution can vary depending on experimental and / or biological settings.Those skilled in the art know how to determine the ideal conditions of the first solution and the second solution.For example, in one embodiment, the magnesium ion concentration of the first solution is higher than 20 mM and / or the magnesium ion concentration of the second solution is lower than 20 mM.

[0042] In some embodiments, the nanostructure may comprise a first portion and a second portion, where the first portion may be movable relative to the second portion. Furthermore, the first portion and the second portion may each comprise one of at least two coupling sites of a coupling site set that may bind to at least one coupling molecule, or the first portion and the second portion may each comprise at least two coupling sites that may bind to at least one coupling molecule. In some preferred embodiments, a "moiety" may be an entity.

[0043] Further, a subset of the set of coupling sites may include at least one coupling site contained by a first portion and at least one coupling site contained by a second portion. That is, each portion may include at least one coupling site. The first portion and the second portion may be movably attached to one another, for example, by a hinge or pivot.

[0044] In general, it should be understood that a nanostructure comprising, for example, a first portion and a second portion should not be interpreted to mean that the nanostructure comprises only one first portion and one second portion. Instead, a nanostructure may also have more than one first portion and more than one second portion. In other words (unless expressly stated or obvious to one of skill in the art), the use of articles (e.g., "a", "an") should not be understood to exclude the plural. For example, a nanostructure comprising two first portions and / or two second portions should also be interpreted as encompassed by "a nanostructure comprising a first portion and a second portion". In some embodiments, a nanostructure may comprise at least one additional portion, i.e., a third, fourth, fifth, sixth, seventh, eighth, or any other number of additional portions.

[0045] It is particularly preferred that the nanostructure comprises at least one molecule selected from the group consisting of nucleic acids, such as DNA, RNA, DNA aptamers, RNA aptamers, or peptide nucleic acids (PNAs), polypeptides, peptides, glycoproteins, peptidomimetics, and antigen-binding constructs, such as antibodies, antibody-like molecules, antigen-binding derivatives, or antigen-binding fragments thereof.

[0046] In a further preferred embodiment, the nanostructure comprises at least one nucleic acid, wherein the nucleic acid has at least one modification, such as a 2'-O-alkyl modification, such as a 2'-O-methoxy-ethyl (MOE) or 2'-O-methyl (OMe) modification, an ethylene-bridged nucleic acid (ENA), a 2'-fluoro N 3 -P 5 The nucleic acid may comprise a chemical modification selected from a modified internucleoside linkage, a modified nucleobase, or a modified sugar moiety, such as a 2'-fluoro (2'-F) nucleic acid, a 1',5'-anhydrohexitol nucleic acid (HNA), or a locked nucleic acid (LNA), such as a 1'-phosphoramidite.

[0047] Yet another embodiment relates to the nanostructure of the present invention, wherein the nanostructure is at least partially formed by a DNA origami structure. The DNA origami structure may include at least one scaffolding strand, i.e., preferably a single-stranded polynucleotide scaffold DNA having a known sequence, and optionally at least one staple strand. The term "DNA origami structure" as used herein refers to a nanostructure that includes at least one scaffolding strand and a plurality of single-stranded oligonucleotide staple strands. The DNA origami structure may be formed by self-assembly, and available software for designing the corresponding scaffolding strand and staple strand may assist in the DNA origami manufacturing technique.

[0048] As used herein, the term "scaffolding strand" refers to a strand that constitutes and / or traverses the main portion of a DNA origami structure and / or a DNA origami subunit ("entity").

[0049] The term "staple strand" as used in this invention refers to a single-stranded oligonucleotide molecule that is at least partially complementary to a scaffolding strand. In general, staple strands can be used, for example, to introduce coupling sites into DNA origami structures and / or DNA origami subunits ("entities").

[0050] The DNA origami structure may include a plurality of single-stranded oligonucleotide staple strands, where each staple strand may be at least partially complementary to at least one scaffolding strand. Further, each of the staple strands may be configured to bind to at least one scaffolding strand in two separate locations, where the at least one scaffolding strand may be folded and / or positioned to form a desired nanostructure.

[0051] A preferred embodiment is a nanostructure of the invention, wherein the nanostructure is at least partially formed by a DNA origami structure, wherein the DNA origami structure comprises at least one scaffolding strand; The DNA origami structure further comprises a plurality of single-stranded oligonucleotide staple strands; Each staple strand is at least partially complementary to at least one scaffolding strand; and Each of the staple strands is configured to bind to at least one of the at least one complementary scaffolding strands, where the at least one scaffolding strand is folded and / or arranged to form a desired nanostructure.

[0052] In a further embodiment, at least one coupling site is present in at least one staple strand.

[0053] Furthermore, in embodiments in which the nanostructure is at least partially formed by a DNA origami structure, the DNA origami structure comprising at least one scaffolding strand and a plurality of staple strands, the molecule may be attached to at least one of the scaffolding strands or one of the staple strands by a linker molecule, where the linker molecule may connect to a portion of the DNA strand that is complementary to a portion of at least one of the scaffolding strands or a portion of the staple strand.

[0054] In those embodiments in which the nanostructure comprises at least one first entity and at least one second entity, the at least one first entity and the at least one second entity may comprise the same shape or distinct shapes. Moreover, the shape may be, for example, triangular, circular, cuboid, i.e., rectangular, or any other shape. Similarly, in those embodiments in which the nanostructure comprises a first portion and a second portion, the first portion and the second portion may comprise the same shape or distinct shapes. The shape may be, for example, triangular, circular, cuboid, i.e., rectangular, or any other shape.

[0055] In some embodiments, at least one coupling site may be a molecule. A preferred embodiment relates to the nanostructure of the invention, in which at least one coupling site is a molecule. In a particularly preferred embodiment, at least one coupling site and / or at least one coupling molecule is a molecule selected from the group consisting of antigens, antigen fragments, antigen mimetics, antibodies, antibody-like molecules, antibody mimetics, antigen-binding derivatives, or antigen-binding fragments, such as antigen-binding constructs, DNA strands, biotin molecules, streptavidin molecules, maleimide-thiol chemistry molecules, click chemistry molecules, SNAP-tag proteins, and CLIP-tag molecules.

[0056] In particularly preferred embodiments, the coupling moiety is an antigen, an antigen fragment, and / or an antigen mimetic.

[0057] In further embodiments, the coupling molecule is an antigen-binding construct, such as an antibody, an antibody-like molecule, an antibody mimetic, an antigen-binding derivative, or an antigen-binding fragment thereof.

[0058] Thus, in one preferred embodiment, the interaction between the coupling site and the coupling molecule is characterized by the specific binding of an antibody-antigen interaction. A person skilled in the art can identify similar specific interactions, which are also intended to be within the scope of this invention. As an example, the specific binding of a streptavidin molecule to a biotin molecule can also be used, for example, the coupling site can be a streptavidin molecule and the coupling molecule can be a biotin molecule, or vice versa. However, a wide variety of interactions can be utilized in such nanostructures, which can be advantageous compared to current technologies that rely on specific and limited interactions. That is, due to the wide range of interactions available, a conditionally switchable nanostructure is provided that is more versatile and flexible compared to the nanostructures disclosed in the prior art.

[0059] Also preferred is where the coupling molecule is an IgG antibody, an IgG antibody fragment, an IgG antibody mimic, an IgM antibody, an IgM antibody fragment, an IgM antibody mimic, an IgD antibody, an IgD antibody fragment, an IgD antibody mimic, an IgA antibody, an IgA antibody fragment, an IgA antibody mimic, an IgE antibody, an IgE antibody fragment, or an IgE antibody mimic, preferably where said antibody, antibody fragment, or antibody mimic comprises two identical antigen binding sites, more preferably where said antigen binding sites bind to said at least two coupling sites of the coupling site set.

[0060] Yet another particularly preferred embodiment relates to the above nanostructure, in which one IgG antibody, one IgG antibody fragment, one IgG antibody mimic, one IgM antibody, one IgM antibody fragment, one IgM antibody mimic, one IgD antibody, one IgD antibody fragment, one IgD antibody mimic, one IgA antibody, one IgA antibody fragment, one IgA antibody mimic, one IgE antibody, one IgE antibody fragment or one IgE antibody mimic is bound to one coupling site set.

[0061] Nanostructures of the invention can be of any length, in preferred embodiments, the nanostructures have a maximum length, and in particularly preferred embodiments, the maximum length is less than 1000 nm, preferably less than 500 nm, such as about 100 nm or less.

[0062] A further aspect of the invention is a system comprising a nanostructure according to the invention, the system comprising: (i) at least one nanostructure comprising at least two coupling sites capable of binding to at least one coupling molecule, the at least two coupling sites being configured as a coupling site set; and (ii) at least one coupling molecule The present invention relates to a system including:

[0063] The term "system" refers to a structure comprising at least one DNA origami structure ("nanostructure") and at least one coupling molecule. In some embodiments, however, the system may comprise multiple coupling molecules.

[0064] In a preferred embodiment, the system comprises: (i) at least one nanostructure comprising at least one first entity and at least one second entity, each of the at least one first entity and the at least one second entity comprising at least one coupling site of at least one coupling site set, preferably, the at least one coupling site on the at least one first entity and the at least one coupling site on the at least one second entity being located on symmetrical positions of the entities; and (ii) at least one coupling molecule, preferably a plurality of coupling molecules; Including, Here, the at least one coupling molecule is configured to couple the at least one first entity of the nanostructure to the at least one second entity of the nanostructure when the at least one coupling molecule is bound to the at least one coupling site set.

[0065] Yet another aspect of the invention, which can be combined with any of the other preferred embodiments and / or aspects of the invention, relates to a method for conditionally switching between different configurations of the nanostructures of the invention, thereby allowing the identification of at least one molecule to be linked to the conformational rearrangement of the nanostructure. The latter may, in certain embodiments, allow the release of therapeutic and / or cargo molecules, which may be contained within cavities and / or holes in the nanostructures of the invention. The method thus allows the nanostructures to specifically release molecules upon identification of a particular molecule.

[0066] In other words, the present invention relates to conditionally switchable nanostructures that can adopt different configurations depending on the coupling of at least a subset of the set of coupling sites contained in the nanostructure, and the accessibility of therapeutic and / or cargo molecules can change depending on the configuration that the nanostructure adopts. For example, the therapeutic and / or cargo molecule can be significantly more accessible in one configuration than the other, and in some cases, it can even be accessible only in one of the two configurations. This can make it possible to allow access to the therapeutic and / or cargo molecule conditional on the coupling of at least a subset of the coupling sites of the set of coupling sites. This can be advantageous, since it can control the release of molecules, such as therapeutic and / or cargo molecules, based on the presence or absence of at least a subset of the respective competing molecules.

[0067] The term "molecule identification" refers to the identification of, for example, antigens, diseased cells, such as tumor cells, etc. Said identification can occur in vivo, ex vivo, or in vitro. In a preferred embodiment, the identification occurs in vivo in an organism, such as a mammal, preferably a human. In another embodiment, the identification occurs in vitro in a sample obtained from an organism, such as a mammal, preferably a human. The nanostructures preferably adopt different configurations upon identifying a particular molecule, such as a competitor molecule, thereby relating the identification of said molecule, such as a competitor molecule, to the conformational rearrangement of the nanostructure. Further included within the scope of this invention is the need to identify an amount of a particular molecule, such as a competitor molecule, that is higher than or corresponds to a certain threshold value of the particular molecule, such as a competitor molecule, for inducing the conformational rearrangement of the nanostructure.

[0068] The term "conformational rearrangement" refers to a rearrangement from one configuration to another, such as from configuration (A) to configuration (B). The nanostructure preferably adopts configuration (B) when at least one competitor molecule competes with at least one coupling molecule and the at least one coupling molecule is not bound to at least one coupling site set. Upon conformational rearrangement, molecules, such as therapeutic and / or cargo molecules, contained within the hollow interior of the nanostructure can be released and / or activated.

[0069] A preferred aspect of this invention, which may be combined with any of the other preferred embodiments and / or aspects of the invention, is a method for relating the identification of at least one molecule to a conformational rearrangement of a nanostructure, said method comprising: (i) providing a nanostructure according to the invention; (ii) providing at least one coupling molecule, providing said nanostructure in at least one first configuration (A) when at least one coupling molecule provided in (ii) is bound to at least one coupling site set of the nanostructure provided in (i); and (iii) identifying the at least one molecule, The at least one molecule is preferably a competitor molecule. Including, the nanostructure adopts at least one second configuration (B) upon identifying the at least one molecule; Thereby, it relates to a method which makes it possible to relate said identification of at least one molecule to a conformational rearrangement of said nanostructure.

[0070] Whenever method steps are listed in this specification and also in the appended claims, it should be noted that the order in which the steps are listed in this document may be accidental. That is, unless otherwise specified or obvious to a person skilled in the art, the order in which the steps are listed may also be different. For example, when this document describes, for example, that a method includes steps (A) and (B), this does not necessarily mean that step (A) precedes step (B), but it is also possible that step (A) is performed (at least partially) simultaneously with step (B) or that step (B) precedes step (A). Furthermore, when it is said that a certain step (X) precedes another step (Z), this does not imply that there is no step between step (X) and (Z). That is, step (X) preceding step (Z) may include not only the situation in which step (X) is performed immediately before step (Z), but also the situation in which step (X) is performed before one or more steps, such as (Y1), (Y2), (Y3), and then step (Z) is performed. Similar considerations apply when using terms such as "after" or "before."

[0071] In a preferred embodiment of the method of relating the identification of at least one molecule to the conformational rearrangement of a nanostructure, the nanostructure comprises at least one first configuration (A) of a molecule or a second nanostructure, preferably wherein the molecule is selected from a therapeutic molecule and / or a cargo molecule.

[0072] The different entities of the nanostructure may be configured to form chambers and / or cavities when the nanostructure is, for example, in a first configuration (A). Furthermore, therapeutic and / or cargo molecules may be attached to the inner surfaces of the different entities of the nanostructure, for example by rods, by direct or indirect association, or by steric means. The therapeutic and / or cargo molecules may be configured to leave the chambers and / or cavities when the nanostructure is in a second configuration (B). The method may include coupling the coupling moieties to the same coupling molecule. Alternatively, the method may include coupling the coupling moieties to separate coupling molecules.

[0073] It is also preferred that when the nanostructure has at least one second configuration (B), a molecule or a second nanostructure, preferably a molecule selected from a therapeutic molecule and / or a cargo molecule, is released and / or activated upon conformational rearrangement of the nanostructure.

[0074] Thus, depending on the binding of the coupling molecule to the coupling site on the nanostructure, the nanostructure can simply switch between different states. This allows for many different applications. In particular, such nanostructures can be used to deliver therapeutic and / or cargo molecules. This can be used, for example, for controlled release of therapeutic and / or cargo molecules upon identification of a particular cell type and / or molecule. Thus, the therapeutic and / or cargo molecule may be accessible in one configuration of the nanostructure but not in a different configuration, which may differ depending on the presence or absence of a particular cell type and / or molecule. This may be particularly useful for applications in which the accessibility of the therapeutic and / or cargo molecule should be conditional on the presence or absence of a particular cell type and / or molecule, which is preferably achieved by the conformational rearrangement of the nanostructure of the present invention.

[0075] Each of the different entities of the nanostructure may include a cavity configured to form a chamber and / or hole when the nanostructure is, for example, in the first configuration (A). Furthermore, the therapeutic and / or cargo molecules may be attached to the inner surface of the different entities of the nanostructure, for example by a rod, by direct or indirect association, or by steric means, where the therapeutic and / or cargo molecules may be configured to leave the cavity when the nanostructure is in the second configuration (B). One skilled in the art will recognize that the rod may be any type of flexible link between the entity and at least one therapeutic and / or cargo molecule.

[0076] In preferred embodiments, the method is an in vivo method, an ex vivo method, an in vitro method, or an in situ method.

[0077] Further embodiments relate to a method of relating the identification of at least one molecule to a conformational rearrangement of a nanostructure, the nanostructure adopting at least one second configuration (B) when the at least one molecule is in competition with at least one coupling molecule and the at least one coupling molecule is not bound to at least one coupling site set, preferably wherein the magnesium ion concentration of the solution is less than 20 mM.

[0078] In a further aspect, the present invention relates to a material comprising a plurality of nanostructures according to the invention or comprising a system as defined above.

[0079] Another aspect of the present invention relates to a composition comprising at least one or more nanostructures according to the invention or comprising a system as defined above.

[0080] A further aspect of the invention, which may be combined with any of the other preferred embodiments and / or aspects of the invention, relates to a substance comprising at least one or more nanostructures or systems as defined above for use as a medicament.

[0081] Yet another aspect of the present invention, which may be combined with any of the other preferred embodiments and / or aspects of the present invention, relates to a composition comprising at least one or more nanostructures according to the present invention or a system as defined above, for use as a medicament.

[0082] Substances and / or compositions for use as medicines may be advantageous since the nanostructures of the invention may enable targeted treatment of diseases, such as targeted drug delivery. For example, the nanostructure(s) according to embodiments of the invention may be used for the release of agents, such as therapeutic and / or cargo molecules, e.g., drugs, contained within the nanostructure. Preferably, such release is targeted or controlled at a defined location and / or time and / or under defined conditions.

[0083] Furthermore, a material comprising at least one or more nanostructures according to the invention or comprising a system as defined above may be for use in the prevention and / or treatment of proliferative diseases, such as cancer, in the prevention and / or treatment of immune disorders, in the prevention and / or treatment of viral diseases, such as human immunodeficiency virus (HIV) infection, in the prevention and / or treatment of blood clotting disorders, in the prevention and / or treatment of metabolic disorders, in the prevention and / or treatment of infectious disorders and / or in the prevention and / or treatment of diabetes.

[0084] In a further preferred embodiment of the invention, the composition comprising at least one or more nanostructures according to the invention or comprising the system as defined above may be for use in the prevention and / or treatment of proliferative diseases, such as cancer, in the prevention and / or treatment of immune disorders, in the prevention and / or treatment of viral diseases, such as human immunodeficiency virus (HIV) infection, in the prevention and / or treatment of blood clotting disorders, in the prevention and / or treatment of metabolic disorders, in the prevention and / or treatment of infectious disorders and / or in the prevention and / or treatment of diabetes.

[0085] A further aspect of the invention, which may be combined with any of the other preferred embodiments and / or aspects of the invention, relates to a material comprising at least one or more nanostructures or systems as defined above for use in a diagnostic method.

[0086] Yet another aspect of the present invention, which can be combined with any of the other preferred embodiments and / or aspects of the present invention, relates to a composition comprising at least one or more nanostructures according to the present invention or a system as defined above, for use in a diagnostic method.

[0087] Another aspect of the invention, which may be combined with any of the other preferred embodiments and / or aspects of the invention, relates to a vector, in particular an expression vector, comprising a sequence encoding a nanostructure, a sequence of a scaffolding strand and / or a sequence of a staple strand, and / or comprising the sequence of said nanostructure, as defined above.

[0088] A "vector" can be any agent capable of delivering or maintaining a nucleic acid in a host cell, including, but not limited to, plasmids (e.g., DNA plasmids), naked nucleic acids, viral vectors, viruses, nucleic acids complexed with one or more polypeptides or other molecules, and nucleic acids immobilized on solid phase particles. Vectors are described in detail below. Vectors can be useful as agents for delivering or maintaining foreign genes and / or proteins in a host cell. A vector can transduce, transfect, or transform a cell, thereby causing the cell to replicate or express a nucleic acid and / or protein other than that native to the cell, or in a manner not native to the cell. A target cell can be a cell maintained under cell culture conditions or in other in vivo embodiments, or can be part of an organism. A vector can include substances that aid in achieving entry of the nucleic acid into the cell, such as viral particles, liposomes, protein coatings, etc. Any method of transferring a nucleic acid into a cell can be used; unless otherwise indicated, the term vector does not imply any particular method of delivering a nucleic acid into a cell or that any particular cell type is the subject of transduction. The present invention is not limited to any particular vector for the delivery or maintenance of any of the nucleic acids of the invention, including, for example, nucleic acids encoding the nanostructures of the invention.

[0089] The term "expression vector" refers to any double-stranded DNA or double-stranded RNA designed to transcribe RNA, e.g., a construct containing at least one promoter operably linked to a downstream gene or coding region of interest (e.g., a cDNA or genomic DNA fragment encoding a protein, or any RNA of interest). Transfection or transformation of the expression construct into a recipient cell allows the cell to express the RNA or protein encoded by the expression construct. The expression construct can be, for example, a genetically engineered plasmid, virus, or artificial chromosome derived from a bacteriophage, adenovirus, retrovirus, poxvirus, or herpesvirus, or further embodiments described below under "expression vectors". The expression construct can replicate in a living cell, or it can be made synthetically. For the purposes of this application, the terms "expression construct", "expression vector", "vector", and "plasmid" are used interchangeably in a general and exemplary sense to demonstrate the application of the invention, and are not intended to limit the invention to a specific type of expression construct. Furthermore, the term expression construct or vector is intended to include the case where the cells utilized for the assay already contain such DNA sequences endogenously. The expression vector preferably contains a promoter sequence operably linked to the nucleic acid.

[0090] The term "encoding" or more simply "coding" refers to the ability of a nucleotide sequence to code for one or more amino acids. The term does not require a start or stop codon. The amino acid sequence may be encoded in any one of six different reading frames provided by the polynucleotide sequence and its complement. The amino acid sequence may be encoded by deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an artificially synthesized polymer similar to DNA or RNA.

[0091] In another aspect, a recombinant cell is also provided that comprises the nanostructure and / or vector as defined above. A "recombinant cell", sometimes also called a "host cell", is any cell that is susceptible to transformation with a nucleic acid. Preferably, the recombinant or host cell of the invention is a plant cell, a bacterial cell, a yeast cell, an insect cell, or a mammalian cell, such as a human cell. Preferred recombinant cells are selected from cells suitable for recombinant expression of the nanostructure of the invention.

[0092] Another aspect of the invention relates to a pharmaceutical composition comprising the nanostructures, systems, compositions, substances, vectors and / or host cells as defined above, together with a pharma- ceutically acceptable carrier and / or excipient.

[0093] A pharmaceutical composition of the invention should be formulated to be compatible with its intended route of administration. In particularly preferred embodiments, examples of routes of administration of the medicaments and / or compounds of the invention include intravenous, intravaginal, oral, intranasal, intrathecal, intraarterial, intradermal, subcutaneous, transdermal (topical), intraventricular, intraparenchymal, intratumor, transmucosal, rectal, bronchial, parenteral administration, and any other clinically / medically accepted method of administration of the medicaments and / or compounds.

[0094] Yet another aspect of the invention, which may be combined with any of the other aspects or specific embodiments of the invention, relates to a method for preventing and / or treating a proliferative disease, such as cancer, an immune disorder, a viral disease, such as human immunodeficiency virus (HIV) infection, a blood clotting disorder, a metabolic disorder, an infectious disorder, and / or diabetes, the method comprising administering to a subject a therapeutically effective amount of a nanostructure, system, compound, substance and / or pharmaceutical composition according to the invention.

[0095] To calculate the amount required to be a "therapeutically effective amount," a skilled artisan and / or physician can use data obtained from cell culture assays and animal studies to formulate a range of dosages for use in humans. The dosage of such compounds and / or pharmaceutical compositions is preferably administered at a dose that is sufficient to induce ED with little or no toxicity. 50 The therapeutically effective dose for any given compound can be estimated initially from cell culture assays. Dosages are within a range of circulating concentrations that include the IC 50 The pharmaceutical compositions can be formulated in animal models to achieve a circulating plasma concentration range that includes the concentration of the test compound that achieves a half-maximal inhibition of symptoms (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0096] The invention further relates to the nanostructures, systems, compounds, materials and / or pharmaceutical compositions of the invention for use in the manufacture of a medicament for the prevention and / or treatment of proliferative diseases such as cancer, immune disorders, viral diseases such as human immunodeficiency virus (HIV) infection, blood clotting disorders, metabolic disorders, infectious diseases and / or diabetes.

[0097] As used herein, the terms "of the invention," "in accordance with the invention," "inventive," and the like are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0098] The term "comprising" as used herein is to be interpreted as embracing both "including" and "consisting of", both meanings being specifically contemplated according to the present invention and therefore being separately disclosed embodiments. As used herein, "and / or" should be taken as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" should be taken as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually described herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature of interest. The terms typically indicate a deviation of ±20%, ±15%, ±10%, and for example ±5% from the indicated numerical value. As would be recognized by a person skilled in the art, the specific deviation of the numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a greater deviation than an artificial or engineered technical effect. As will be understood by one of ordinary skill in the art, the specific such bias for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have a greater such bias than man-made or engineered technical effects. When an indefinite or definite article, such as "a," "an," or "the," is used when referring to a singular noun, this includes a plural of that noun unless something else is specifically stated.

[0099] All numerical values ​​are considered to be modified by the term "about" in this specification, whether or not it is explicitly stated. The term "about" generally refers to a range of numbers that a person skilled in the art would consider equivalent to the recited value (i.e., have the same function or result). In many cases, the term "about" may include numbers that are rounded to the nearest significant figure. In a particularly preferred embodiment of the present invention, the term "about" may refer to a numerical deviation of up to 20% of the respective numerical value, however, more preferably 15%, 10%, 5%, even more preferably 4%, 3%, 2%, and most preferably 1%. However, whenever a relative term such as "about", "substantially" or "approximately" is used in this specification, such a term should also be interpreted as including the exact term. That is, for example, "substantially one" should also be interpreted as including "(exactly) one".

[0100] It should be understood that application of the teachings of the present invention to a particular problem and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments) will be within the capabilities of one of ordinary skill in the art in light of the teachings contained herein.Unless the context dictates otherwise, the description and definitions of features set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0101] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety. EXAMPLES

[0102] Certain aspects and embodiments of the present invention will now be illustrated, by way of examples, with reference to the description, figures and tables set forth herein. Such examples of methods, uses and other aspects of the present invention are representative only and should not be taken to limit the scope of the present invention to only such representative examples.

[0103] The example shows:

[0104] Example 1: IgG-mediated stabilization and antigen-induced disassembly of icosahedral DNA origami shells and antigen-induced agent release This invention is based on a general strategy for the rearrangement of DNA origami nanoparticles gated by antigen-induced logic. The design principles of this technique are modular in nature and can be applied to any dynamic DNA origami system that can adopt two or more conformational states.

[0105] FIG. 1 shows components used in some embodiments of the present technology to illustrate the notation used herein. More specifically, FIG. 1 shows a nanostructure that can take different configurations (A) and (B), which can also be referred to as closed configuration (A) and open configuration (B). The three-dimensional nanostructure depicted in FIG. 1 can be realized using DNA origami, i.e., by combining scaffolding strands and staple strands to form the required parts and entire devices. Such design can be performed, for example, using software such as caDNAno. That is, a nanostructure containing multiple entities can be made from one scaffolding strand in some embodiments, while in other embodiments, the entities of the nanostructure can be constructed using multiple scaffolding strands.

[0106] The nanostructure is preferably configured to adopt a first configuration (A) when each coupling site of the set of coupling sites, or at least a subset of the coupling sites, is coupled by its respective coupling molecule, and to adopt a second configuration (B) when none of the coupling sites, or only a smaller subset of the coupling sites, are coupled by their respective coupling molecules.

[0107] In the embodiment depicted in Figure 1, the coupling sites are of the same configuration, i.e. they are configured to couple to coupling molecules of the same design, and the nanostructures are of a bivalent nature, i.e. nanostructures with one type of coupling site. However, as already mentioned above, this is not necessary. In some embodiments of the invention, the coupling sites may be attached to separate coupling molecules. The latter refers to multivalent nanostructures, which are structures with multiple different coupling sites.

[0108] The proposed mechanism depicted in Figure 1 is based on two design principles. First, we used the highly conserved structure of IgG antibodies, which bivalently display two identical antigen-binding sites. Taking advantage of this bivalent feature, the antibodies are used as molecular bridges to fix DNA origami nanoparticles in a predefined configuration dictated by strategically placed antigens (fragments) on the DNA origami subunits. Second, antigen-induced activation or rearrangement of the "spring-loaded" nanoparticles is initiated by the presence of soluble antigens that displace the "antibody bridge" from the nanoparticle.

[0109] In the example of FIG. 1, configuration (A) is a closed configuration (such as a "cage"-like structure, e.g., in this particular embodiment, a capsid-like structure assembled from 20 identical DNA origami subunits (triangles) forming an icosahedral DNA origami shell). Configuration (B) is an open configuration, where the DNA origami subunits (triangles) are not coupled via antibody bridges. Thus, a single triangle is visible in the negative stain TEM image of configuration (B).

[0110] As a proof of concept, Figure 1 shows the antibody-mediated spring-loading and subsequent antigen-triggered disassembly of a T=1 capsid-like structure (icosahedral DNA origami shell) self-assembled from 20 identical multilayered DNA origami triangles (Figure 1). To direct antibody binding, "coupling sites" (antigen fragments, e.g., digoxigenin ligands) are introduced at symmetric positions on the sides of each triangle via chemically modified staple oligonucleotides. After self-assembly of the icosahedral shell (T=1 capsid), governed by activation of shape-complementary protrusions and depressions on the triangular interface in shell-favorable buffer conditions ([Mg2+]>20 mM), anti-digoxigenin antibodies are added to crosslink individual triangular subunits. Subsequent reduction of the Mg2+ concentration to inactivate stacking contacts results in spring-loading of the capsid assembly. Finally, the addition of a co-antigen (a "competing antigen", e.g., a soluble digoxigenin antigen) induces disassembly of the capsid by a competitive reaction that displaces the antibody from the DNA-origami-attached antigen (Figure 1). Antigen-induced disassembly of the icosahedral shell occurs at a certain antigen threshold (EC 50 The binding of antibodies occurs at approximately 1 μM, depending on the number of antibodies attached per subunit interface. This concept can be further explored by placing specific combinations of antibodies, creating sophisticated molecular logic operations, or tailoring dose-response curves.

[0111] While the embodiment of Figure 1 is based on a particle self-assembled from multiple components as a proof of concept, the same concept can be applied to actuate single molecular objects such as box-like structures or switches in applications including, but not limited to, molecular diagnostics, logic-gated therapy or drug delivery vehicles. Finally, given the highly conserved bivalent nature of, for example, IgG antibodies and the ease of generating antibodies against virtually any molecular target, this invention could be used to identify a wide variety of antigens, small molecules, peptides and proteins.

[0112] In contrast to current modules that interface DNA nanostructures with antigen-antibody recognition elements, all recognition elements of the nanostructure depicted in FIG. 1 are integrated on the nanostructure itself, rather than relying on diffusive interactions of multiple components. This makes our concept insensitive to dilution effects, a crucial requirement for potential future biomedical applications. Importantly, our design principle is not limited to multicomponent systems whose self-assembly is mediated by shape-complementary stacking contacts. Other strategies, such as temperature- or strand-displacement-dependent complementary overhangs, pH-dependent triplexes or i-motifs, can be envisioned to direct the self-assembly of the nanostructures of this invention and the subsequent spring-loading of the structures by antibody-enhanced interfaces, and are also intended to be within the scope of this invention.

[0113] A concrete example of the mechanism of FIG. 1 and its exemplary use for the release of agents / entities from nanostructures is shown in FIG. 4, which shows antigen-triggered release of viral payload. (a) Scheme showing (from left to right) functionalization of a subset of triangles with Hepatitis B virus core capsid, where binding of HBV particles is mediated by specific antibodies at the inward-facing surface of the triangular shell; assembly of the complete shell with HBV core capsid in the lumen; stapling by anti-Dig IgG antibodies and spring loading by lowering ionic strength; antigen-triggered shell rupture and release of viral payload by adding soluble digoxigenin antigen. Not shown are intermediate steps that detach the HBV-binding antibodies located in the interior of the shell from the shell, leaving the HBV particles free floating in the shell. (b, c, d) Functionalization of a subset of triangles with Hepatitis B virus core capsid in the lumen, where binding of HBV particles is mediated by specific antibodies at the inward-facing surface of the triangular shell; assembly of the complete shell with HBV core capsid in the lumen; stapling by anti-Dig IgG antibodies and spring loading by lowering ionic strength; antigen-triggered shell rupture and release of viral payload by adding soluble digoxigenin antigen. Not shown are intermediate steps that detach the HBV-binding antibodies located in the shell from the shell, leaving the HBV particles free floating in the shell. (b, c, d) Functionalization of a subset of triangles with Hepatitis B virus core capsid in the lumen, where binding of HBV particles is mediated by specific antibodies at the inward-facing surface of the triangular shell; (c, d) Functionalization of a subset of triangles with Hepatitis B virus core capsid in the lumen, where binding of HBV particles is mediated by specific antibodies at the inward-facing surface of the triangular shell; (d) Functionalization of a subset of triangles with Hepatitis B virus core caps 2 Shells assembled under spring-loaded conditions (12 mM MgCl 2 Negative staining TEM images of anti-Dig stapled shells loaded with viral payload in (b,c) and disassembled by adding soluble digoxigenin antigen, leading to the release of the viral payload. Scale bars in (b,c) are 50 nm and in (d) are 100 nm.

[0114] Example 2: Structural characterization of antigen-induced disassembly of the icosahedral shell To structurally characterize and evaluate the Mg2+-dependent IgG-mediated shell stabilization, we performed cryo-EM and laser scanning fluorescence microscopy. 2 An exemplary Cryo-EM micrograph of an icosahedral shell in free-standing ice with anti-digoxigenin antibodies bridging the triangle-triangle interfaces is shown in Figure 2(a), top. A Cryo-EM reconstruction of an assembled icosahedral shell with two anti-digoxigenin antibodies bridging each triangle-triangle interface is shown in Figure 2(a), bottom.

[0115] We then performed laser scanning microscopy. Figure 2e) shows the results for the 8, 12, 16 and 20 mM MgCl 2 The assembled shells were then washed with 0.5x TBE buffer supplemented with 25 mM MgCl. 2 The mice were incubated with or without anti-digoxigenin antibody at 100°C, followed by incubation with each of the MgCl 2 The samples were diluted to 100% concentration and electrophoresed on the corresponding gels. We further characterized configurations (A) and (B) using TEM. The samples were incubated without (left) or with (right) anti-digoxigenin antibody and treated with 12 mM MgCl. 2 Negative staining TEM images of shells diluted to 100 μg / ml and incubated without and with anti-digoxigenin antibody followed by various final MgCl 2 The normalized FRET ratios of shells diluted to concentrations are shown in Figure 2(e). Each side of the triangular subunit is functionalized with Cy3 and Cy5 fluorophores at symmetrical positions, which results in energy transfer only when the shell is assembled. 2The initial decrease in FRET ratio with concentration is caused by shell swelling due to increasing electrostatic repulsion. To further characterize antigen-induced disassembly of icosahedral shells, we used laser scanning microscopy to demonstrate antigen-induced disassembly of icosahedral shells. Figure 3(a) shows that addition of soluble digoxigenin ligands that compete for binding with the shell-stabilizing anti-digoxigenin antibodies displaces the antibodies from the spring-loaded shells, which in turn induces shell disassembly at destabilizing Mg2+ ion concentrations. Assembled shells with anti-digoxigenin antibodies were incubated with increasing concentrations of digoxigenin to demonstrate that anti-digoxigenin-stabilized shells (25 μM soluble digoxigenin) disassemble at increasing concentrations of soluble digoxigenin antigen, as shown in Figure 3(a). Figure 3(b) shows disassembly of anti-dinitrophenol-stabilized shells at increasing concentrations of soluble 2,4-dinitrophenol ligands.

[0116] Figure 3(f) shows, in the left part, that shell disassembly gated by AND gate logic is realized by placing both digoxigenin and 2,4-dinitrophenol ligands and their respective antibodies on the triangle-triangle interface. AND-gated shell disassembly therefore requires the presence of both soluble digoxigenin and 2,4-dinitrophenol. Figure 3(f) shows, in the right part, that shell disassembly gated by AND gate logic is realized by placing both digoxigenin and 2,4-dinitrophenol ligands and their respective antibodies on the triangle-triangle interface. 2 Laser-scanned fluorescent images of 0.75% agarose gel in 0.5x TBE buffer supplemented with 100% DMSO. Assembled AND gate shells with anti-digoxigenin and anti-dinitrophenol antibodies were incubated with increasing concentrations of Top: Digoxigenin, Middle: 2,4-dinitrophenol, and Bottom: Digoxigenin and 2,4-dinitrophenol. Negative stain TEM images of shells with anti-digoxigenin and anti-dinitrophenol incubated with Top: 25 μM Digoxigenin, Middle: 5 mM 2,4-dinitrophenol, and Bottom: 25 μM Digoxigenin and 5 mM 2,4-dinitrophenol.

[0117] Although the preferred embodiment has been described above with reference to the accompanying drawings, those skilled in the art will understand that the embodiment is provided for illustrative purposes only and should not be construed in any way as limiting the scope of the present invention, which is defined by the claims.

Claims

1. A nanostructure comprising at least two coupling sites, the at least two coupling sites capable of binding to at least one coupling molecule, the at least two coupling sites being configured as a coupling site set, and optionally, one coupling site set being configured to bind to one coupling molecule.

2. 2. The nanostructure of claim 1, wherein the nanostructure comprises at least one first entity and at least one second entity, each of the at least one first entity and the at least one second entity comprising at least one coupling site of at least one coupling site set, preferably the at least one coupling site of the at least one first entity and the at least one coupling site of the at least one second entity being located on symmetrical positions of the at least one first entity and the at least one second entity, and optionally the at least one coupling molecule configured to couple the at least one first entity of the nanostructure to the at least one second entity of the nanostructure when the at least one coupling molecule is bound to the at least one coupling site set.

3. The nanostructures may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any number of the at least one first entity, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any number of the at least one second entity.

3. The nanostructure of claim 1 or 2, comprising a plurality of said at least one first entity and a plurality of said at least one second entity, such as one or more second entities, and optionally wherein the nanostructure comprises a plurality of coupling site sets, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or any number of coupling site sets, preferably each coupling site set consisting of two coupling sites.

4. 3. The nanostructure of claim 1 or 2, wherein the nanostructure is configured to have at least one first configuration (A), and at least one second configuration (B), optionally at least one third configuration (C), at least one fourth configuration (D), or any other number of configurations.

5. 5. The nanostructure of claim 4, wherein the nanostructure is configured to adopt at least one first configuration (A) when at least one coupling molecule is bound to at least one of the set of coupling sites, and / or the nanostructure is configured to adopt at least one second configuration (B) when at least one coupling molecule is not bound to at least one of the set of coupling sites, and optionally the nanostructure is configured to adopt at least one first configuration (A) when little or no at least one competitor molecule is present, and / or the nanostructure is configured to adopt at least one second configuration (B) when a greater amount of at least one competitor molecule is present.

6. 5. The nanostructure of claim 4, wherein the nanostructure is configured to adopt at least one first configuration (A) in a first solution, the first solution preferably comprising a higher concentration of magnesium ions compared to a second solution, and / or the nanostructure is configured to adopt at least one second configuration (B) in a second solution, the second solution preferably comprising a lower concentration of magnesium ions compared to the first solution, optionally wherein the magnesium ion concentration of the first solution is higher than 20 mM and / or wherein the magnesium ion concentration of the second solution is lower than 20 mM.

7. 3. The nanostructure of claim 1 or 2, wherein the nanostructure comprises at least one molecule selected from the group consisting of nucleic acids, such as DNA, RNA, DNA aptamers, RNA aptamers, or peptide nucleic acids (PNAs), polypeptides, peptides, glycoproteins, peptidomimetics, and antigen-binding constructs, such as antibodies, antibody-like molecules, antigen-binding derivatives, or antigen-binding fragments thereof.

8. the nanostructure is at least partially formed by a DNA origami structure; the DNA origami structure comprises at least one scaffolding strand; the DNA origami structure further comprises a plurality of single-stranded oligonucleotide staple strands; Each staple strand is at least partially complementary to at least one scaffolding strand; and 3. The nanostructure of claim 1 or 2, wherein each of the staple strands is configured to bind to at least one of the at least one scaffolding strands, and wherein the at least one scaffolding strand is folded and / or arranged to form a desired nanostructure.

9. 3. The nanostructure according to claim 1 or 2, wherein at least one coupling site is a molecule, preferably said at least one coupling site and / or at least one coupling molecule is a molecule selected from the group consisting of an antigen, an antigen-binding construct such as an antigen, an antigen-fragment, an antigen-mimetic, an antibody, an antibody-like molecule, an antibody-mimetic, an antigen-binding derivative or an antigen-binding fragment, a DNA strand, a biotin molecule, a streptavidin molecule, a maleimide-thiol chemistry molecule, a click chemistry molecule, a SNAP-tag protein and a CLIP-tag molecule, preferably said coupling molecule is an antigen-binding construct such as an antibody, an antibody-like molecule, an antibody-mimetic, an antigen-binding derivative or an antigen-binding fragment thereof, and said coupling site is an antigen, an antigen-fragment and / or an antigen-mimetic.

10. 3. The nanostructure of claim 1 or 2, wherein the coupling molecule is an IgG antibody, an IgG antibody fragment, an IgG antibody mimic, an IgM antibody, an IgM antibody fragment, an IgM antibody mimic, an IgD antibody, an IgD antibody fragment, an IgD antibody mimic, an IgA antibody, an IgA antibody fragment, an IgA antibody mimic, an IgE antibody, an IgE antibody fragment, or an IgE antibody mimic, preferably said antibody, antibody fragment, or antibody mimic comprises two identical antigen binding sites, more preferably said antigen binding sites bind to said at least two coupling sites of the coupling site set.

11. 3. The nanostructure of claim 1 or 2, wherein the nanostructure comprises a maximum length, the maximum length being smaller than 1000 nm, preferably smaller than 500 nm, such as 100 nm.

12. A system comprising the nanostructure of claim 1 or 2, comprising: (i) at least one nanostructure comprising at least two coupling sites, the at least two coupling sites being capable of binding to at least one coupling molecule, the at least two coupling sites being configured as a coupling site set; and (ii) at least one coupling molecule Including, the system.

13. The system, at least one nanostructure comprising at least one first entity and at least one second entity, each of said at least one first entity and said at least one second entity comprising at least one coupling site of at least one coupling site set, preferably said at least one coupling site on said at least one first entity and said at least one coupling site on said at least one second entity being located on symmetrical positions of said entities; and At least one coupling molecule, preferably a plurality of coupling molecules Including, 13. The system of claim 12, wherein the at least one coupling molecule is configured to couple the at least one first entity of the nanostructure to the at least one second entity of the nanostructure when the at least one coupling molecule is bound to the at least one coupling site set.

14. 1. A method for relating identification of at least one molecule to a conformational rearrangement of a nanostructure, the method comprising: (i) providing a nanostructure according to claim 1 or 2; (ii) providing at least one coupling molecule, providing, when at least one coupling molecule provided in (ii) is bound to at least one coupling site set of the nanostructure provided in (i), the nanostructure adopts at least one first configuration (A); and (iii) identifying the at least one molecule. Including, wherein said at least one molecule is preferably a competitor molecule; the nanostructure assumes at least one second configuration (B) upon identifying the at least one molecule; thereby allowing said identification of at least one molecule to be related to a conformational rearrangement of said nanostructure; Optionally, the nanostructure surrounds the at least one molecule in the first configuration (A) or the second nanostructure, preferably the molecule is selected from a therapeutic molecule and / or a cargo molecule; Further optionally, the molecule or the second nanostructure, preferably the molecule selected from a therapeutic molecule and / or a cargo molecule, is released and / or activated upon conformational rearrangement of the nanostructure when the nanostructure is in at least one second configuration (B).

15. A composition comprising at least one or more nanostructures according to claim 1 or 2 for use as a medicine and / or for use in a diagnostic method.

16. A composition comprising the system described in claim 12 for use as a medicine and / or for use in a diagnostic method.

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