Method to impose a geometric arrangement to binding partners present on a biological interface

Abstract

The present invention introduces a method for enhancing the selectivity of molecular interactions on biological interfaces by imposing a spatial geometry on nanoscale supports, referred to as nanostructures These nanostructures, designed as two- or three-dimensional platforms, comprise binding units present on the same plane with predetermined set angles or distances. The binding units, identical or of different identities, are strategically arranged to restrict spatial mobility. The method involves placing the nanostructures in contact with corresponding binding partners, allowing the organization of binding partners in the imposed spatial geometry. The invention provides a versatile approach applicable to various biological systems, offering precise control over molecular interactions at the nanoscale. Applications include targeted drug delivery, diagnostic assays, and molecular engineering.

Description

Method to impose a geometric arrangement to binding partners present on a biological interfaceField of the Invention

[0001] The present invention relates to the field of biotechnology, specifically to methods for imposing a geometric arrangement to binding partners present on a biological interface. More particularly, the invention pertains to the imposition of spatial geometries on one or multiple components present on a biological interface, thereby confining the mobility of interacting partners in space to facilitate precise and selective binding events and up- or down-regulate cell reactions.Background of the Invention

[0002] Multivalent affinity enhancement has been a widely recognized strategy in biotechnology for several decades. This approach capitalizes on the principle that the simultaneous engagement of multiple binding units can significantly augment the affinity and specificity of interactions between molecular entities.

[0003] The field of nucleic acid nanotechnology has over the past several decades developed technologies enabling the fabrication of programmable DNA-based assemblies of prescribed size, geometry, rigidity, and chemical Composition (Jun, et al., Nucleic Acids Research, 49 (18), 10265-10274 (2021), Pettersen, et al., Journal of Computational Chemistry, 25 (13), 1605-1612 (2004), Benson, et al., Angewandte Chemie International Edition, 55 (31), 8869-8872 (2016), Jun, et al., ACS Nano (2019), Benson, et al., Nature, 523 (7561), 441-444 (2016), Dietz, et al., Science, 325 (5941), 725-730 (2009), Castro, et al., Nature Methods, 8 (3), 221-229 (2011), Douglas, et al., Nucleic Acids Research, 37 (15), 5001-5006 (2009), Veneziano, et al., Science, 352 (6293), 1534-1534 (2016)). These nanomaterials now represent a toolbox for the design and fabrication of nanodevices capable of interacting with diverse cellular environments (Veneziano, et al., Nature Nanotechnology, 15 (8), 716-723 (2020), Lee, et al., Nature Nanotechnology, 7 (6), 389-393 (2012)). One approach to designing nucleic acid nanostructures on the 10-100 nm scale is the concept of DNA origami, wherein programmed regions of a long, single-stranded DNA scaffold that are far apart in sequence space are brought into spatial proximity through the hybridization of small, single-stranded DNA staples.

[0004] However, the conventional multivalent strategies predominantly focus on high valency interactions, overlooking the potential for enhanced selectivity at lower valency,especially when operating at the nanoscale. This uncharted territory represents a critical frontier in molecular engineering, demanding innovative approaches to harness the full potential of affinity enhancement.

[0005] The present invention addresses this need by introducing a novel method that leverages spatial control over the arrangement of binding units on a nanostructure. By using planar rigid nanostructure(s) at the nanoscale, comprising two or more binding units with specific geometric arrangements, the invention restricts the spatial freedom of binding partners. This geometric confinement enhances the precision and selectivity of interactions on biological interfaces and allows to up- or down-regulate specific cell reactions at will.

[0006] It was shown that fixed CpG dimer pairs at distances of 7 nm or 38 nm on a 2D nano-disk showed that the 7 nm dimer pair, which matched the distance between binding sites in the TLR9 dimer, induced increased immune activation, demonstrating the important of inter-ligand distance (Comberlato, et al., Nano Letters (2022), however the present inventors go even further by demonstrating that the specific patterns and shapes created by binding units on the surface of a nanostructure have more influence than distance between binding sites.Summary of the Invention

[0007] The present invention introduces a novel method for achieving high precision and selectivity in molecular interactions on biological interfaces. The innovation lies in the imposition of spatial geometries on nanostructures, thereby limiting the mobility of interacting partners and enabling enhanced selectivity in binding events.

[0008] At the core of this method is the provision of nanostructures, which take the form of planar and rigid structures. These nanostructures are engineered to incorporate two or more binding units, each possessing a specific location on the nanostructures and angles and distances to each other on the nanostructures, thereby imposing a defined spatial framework upon the interacting partners.

[0009] The introduction of this geometric confinement represents a paradigm shift from conventional multivalent strategies which focus on the overall strength of the interaction. The present invention focuses on the impaired mobility upon binding, only possible when binding units are constrained in their motion. Thus, by restricting the spatial freedom of the binding units and their respective ligands on the biological interface, the invention imparts a newfound level of control over molecular interactions. This precision extendsto both the timing and orientation of binding events, leading to high levels of selectivity, even at low valency.

[0010] Applications of this method span a wide array of fields within biotechnology. From targeted drug delivery and precision medicine to the development of highly specific diagnostic assays, the potential impact is vast and transformative with implications for the design of novel therapeutics, biomaterials, and biosensors.Detailed Description of the Invention

[0011] The present invention firstly relates to a method to impose a geometric arrangement to one or multiple binding partners present on a biological interface comprising the steps of: a) providing planar rigid nanostructure(s) comprising two or more copies of one or more binding units of the same or of different identity bound to the surface of the nanostructure;Wherein said binding units comprise set angles and set distance(s) between adjacent binding units and / or a set position on the surface of the nanostructure; and b) putting the nanostructure(s) in contact with the binding units’ corresponding binding partners to allow the organization of the binding partners in the imposed geometric arrangement.

[0012] In other words, this method imposes a restricted spatial freedom upon the binding units, ensuring precise control over their interactions with complementary binding partners on the biological interface.

[0013] What is meant herein by “geometric arrangement” is the specific arrangement and orientation of binding partners present on a biological interface which can be defined by set angles or distances to each other and / or the position of each binding unit on the surface of the nanostructure. In the present invention, it involves the precise positioning of binding units in relation to each other on a planar rigid nanostructure, forcing therefore the binding partners of said binding units into a specific geometric arrangement upon binding with the binding units.

[0014] The term “antigen” as used herein is defined as a molecule capable of being recognized or bound by an antibody, B-cell receptor or T-cell receptor. An “immunogen” is an antigen that is additionally capable of provoking an immune response against itself (e.g., upon administration to a mammal, optionally in conjunction with an adjuvant). This immune response can involve either antibody production, or the activation of specific immunologically-competent cells, or both. Any macromolecule, including virtually allproteins or peptides as well as lipids and oligo- and polysaccharides, can serve as an antigen or immunogen. Furthermore, antigens / immunogens can be derived from recombinant or genomic DNA. Any DNA that includes a nucleotide sequence or a partial nucleotide sequence encoding a protein or peptide that elicits an immune response therefore encodes an “immunogen” as that term is used herein. An antigen / immunogen need not be encoded solely by a full-length nucleotide sequence of a gene. An antigen / immunogen need not be encoded by a “gene” at all. An antigen / immunogen can be generated, synthesized, or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0015] Each binding partner has a unique molecular structure or sequence that allows it to selectively interact with its complementary partner.

[0016] What is meant herein by “binding units” is a molecular entity or component that is capable of selectively interacting with a complementary molecule on a biological interface, it can also be interchangeably called “binder”, “binding agent”, “active moiety” or “targeting moiety” or “targeting molecule”.

[0017] The binding unit(s) are bound to the surface of the nanostructure (i.e immobilized on the surface of the nanostructure), preferably covalently bound to the surface of the nanostructure.

[0018] The binding unit may be a specific sequence of nucleotides or a specific chemical moiety. It can include molecules like ligands, receptors, or any other molecular entity that exhibits selective binding affinity for its complementary partner, for example, peptides, proteins, nucleic acids, lipids, and / or polysaccharides. In preferred embodiments, the binding unit is a Peptide, adhesion peptide, protein, immune checkpoint protein, antibody or antibody fragment, aptamer, natural or modified nucleic acid sequence. The binding unit can be antigens associated with one or more diseases or conditions including, but not limited to, infectious diseases, autoimmune diseases, and cancer.

[0019] In some embodiments, the binding units can be molecules interacting with binding partners (or target) selected from the group consisting of Integrin family, Frizzled, Lrp 5 / 6, Lgr5, Znrf3, BMPR family, TGF family, INSR I IGF1 family, EGFR family, ErbB family, and combination thereof.

[0020] In other embodiments, the binding units can be molecules interacting with binding partners (or target) from the integrin family, preferably fragments of collagen, fibronectin, hyaluronic acid, laminin and their subtypes.

[0021] In some embodiments, the binding units can be molecules interacting with binding partners (or target) selected from the group consisting of MHC-I, MHC-II, PD-L1 , PD-L2, CD80, CD86, and combination thereof.

[0022] The terms “polypeptide,” “peptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids.

[0023] What is meant herein by “binding partners” is the molecules or molecular entities that interact with each other through specific chemical or physical forces. These interactions can include processes like binding, attachment, or association. The term "binding partners" is a broad and inclusive term used to describe any molecules involved in a specific binding event. It can encompass both the molecule that is immobilized on the nanostructure (binding unit) and the molecule that interacts with it (ligand or receptor), depending on the specific context of the interaction.

[0024] The “binding partners” can be selected in the group consisting of Antibody- Antigen wherein the antibodies also comprise nanobodies and any synthetically derived antibody fragments, Enzyme-Substrate, Receptor-Ligand, DNA-Protein, Hormone- Receptor, RNA-Molecule, Ion-Channel, Coenzyme-Enzyme, Drug- Receptor, RNA-DNA, Nucleic Acid-Protein, Cell Adhesion Molecules, small Molecule-carbohydrate and combinations therein.

[0025] In the context of the invention, "biological interface" (also called “biointerface”) refers to the region or surface where specific molecular interactions occur within a biological system, more specifically the region of contact between a biomolecule, cell, biological tissue or living organism or organic material considered living with another biomaterial or inorganic / organic material. It represents the interface or boundary where two or more biological components come into contact and engage in binding events. This term encompasses various scenarios, depending on the specific application of the invention. It could refer to interactions between molecules within a cell, between cells, or between a biological entity and an external substance or environment. For instance, in a cellular context, the biological interface could be the region of contact between thenanostructure surface and the cell membrane, where binding partners interact (e.g receptors interacting with ligands).

[0026] In the context of the invention, a "nanostructure" refers to an extremely small- scale object or entity, typically at the nanometer (nm) scale. It is a deliberately engineered structure designed to have specific properties or characteristics at this exceptionally small size. In the present invention, a nanostructure is a support or scaffold created at the nanoscale, potentially using techniques like DNA origami. This nanostructure serves as a platform for arranging binding units with defined spatial relationships. The nanostructure's dimensions, which are smaller than 100 nanometers, are crucial for achieving precise spatial control over molecular interactions. Overall, the term "nanostructure" emphasizes the deliberate and highly controlled engineering of a structure at the nanoscale to facilitate specific molecular interactions on biological interfaces.

[0027] In the context of the invention, a “planar” nanostructure refers to a structure that primarily extends in two spatial dimensions, typically along length and width. It can be visualized as having a flat or planar shape. In other words, a planar nanostructure is one that lies predominantly within a single plane. In this configuration, the binding units are arranged within the same plane of the nanostructure in order to orient all binding units towards the same biological interface.

[0028] In some embodiments, the nanostructure has a low curvature at the nanometer scale such that all binding units have equal or close z-position on the nanostructure. As used herein, the term "low curvature" means that the nanostructure has a surface that is not significantly curved or bent at the nanometer scale. It implies that any curvature present in the nanostructure does not deviate significantly from a flat, planar configuration, preferably does not deviate more than between 5% and 10%, preferably between 2% and 5%.

[0029] As used herein, the term “rigid” refers to the property of the nanostructure to maintain its shape and structural integrity in solution and with or without applied forces or stresses. A rigid nanostructure is one that does not deform or change its spatial arrangement easily when subjected to external pressures, movements, or binding events. The term “rigid” is known and understood in the field of nanostructure, see for instance WO2017189870 or Ohtsuki, Shozo et al. “Folding of single-stranded circular DNA into rigid rectangular DNA accelerates its cellular uptake.” Nanoscale (2019).

[0030] In the context of this invention, the rigidity of the nanostructure ensures that the defined geometric arrangement, which is crucial in molecular interactions, remains intact. A rigid nanostructure effectively imposes and maintains the desired geometric arrangement of binding units, restricting the mobility of interacting partners on the biological interface.

[0031] The rigidity of the nanostructure can be routinely achieved by modelization of the nanostructure with molecular simulations and the plotting of the probability distribution function of the spatial freedom of the extremities (Do-Nyun Kim, Fabian Kilchherr, Hendrik Dietz, Mark Bathe, Quantitative prediction of 3D solution shape and flexibility of nucleic acid nanostructures, Nucleic Acids Research, Volume 40, Issue 7, 1 April 2012, Pages 2862-2868). In the case of a DNA based nanostructure, the structural rigidity can be calculated using any software that calculates the RMSF (root mean square fluctuations) for example the software tool CaNDo. CanDo utilizes a simplified model of DNA optimized for the DNA origami simulation intended to predict an equilibrium shape of the origami and estimate its mobility. RMSF (root mean square fluctuations) is given in nm from the average and is the standard way in the art to define rigidity of a nanostructure. An RMSF inferior to 5nm, preferably under 2.5nm and more preferably under 1nm is considered sufficiently rigid. A DNA origami disk according to the present invention has preferably a RMSF under 0.8nm. As such, in some embodiments, the nanostructure according to the invention has a RMSF inferior to 5nm, more preferably under 2.5nm, more preferably under 1nm and even more preferably under 0,8nm.

[0032] Because the nanostructure is rigid, the mean and median spacing between two adjacent binding units cannot significantly change (preferably less than 10%, more preferably less than 5%, even more preferably less than 1%). The binding units present on the nanostructure according to the invention have thus limited spatial freedom and mobility in order to restrict the mobility of the interacting partner in space during the interaction.

[0033] In some embodiments, the nanostructure(s) according to the invention are produced using DNA origami, a technique known for its precision and versatility in creating nanoscale structures. Preferably, the nanostructure(s) is a nucleic acid nanostructure of arbitrary 2-D or 3-D shape and / or a nanoplatform.

[0034] The terms “scaffolded origami”, “origami”, “nucleic acid nanoparticle”, “nucleic acid nanostructure”” “nucleic acid assembly” are used interchangeably. They can be one or more short single strands of nucleic acids (staple strands) (e.g., DNA) that fold a long,single strand of polynucleotide (scaffold strand) into desired shapes on the order of about 10 nm to a micron, or more. Wireframe scaffolded DNA origami may use edges having 2, 4, 6, or more duplexes crosslinked in parallel to endow rigidity to the nanoparticle (Jun et al., ACS Nano, 2019, 10.1021 / acsnano.8b08671 ; Veneziano et al., Science, 352(6293): 1534 (2016)). Single-stranded DNA scaffold may be produced from M13 or using a helper plasmid as shown by Shepherd, et al., bioRxiv 521443 (2019), doi: https: / / doi.org / 10.1101 / 521443 and Praetorius et al., Nature, 552:84-87 (2017). Alternatively, single-stranded synthetic nucleic acid can fold into an origami object without helper strands, for example, using parallel or paranemic crossover motifs. Alternatively, purely staple strands can form nucleic acid memory blocks of finite extent. The scaffolded origami or origami can be composed of deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs or modified nucleotides thereof, including, but not limited to locked nucleic acids (LNA) and peptide nucleic acids (PNA). A scaffold or origami composed of DNA can be referred to as, for example a scaffolded DNA origami or DNA origami, etc. It will be appreciated that where compositions, methods, and systems herein are discussed or exemplified with DNA (e.g., DNA origami), other nucleic acid molecules can be substituted. The scaffold can be a two-dimensional or three- dimensional shape selected from the group consisting of a sheet, square, rectangle, nanotube, cylinder, ring, disc, ribbon, box, cube, pyramide cross and rod. The DNA scaffold can be assembled by a single- stranded DNA backbone chain and / or at least 50 single-stranded DNA staple chains. DNA-origami nanostructures or nucleic acid nanostructures can be produced for instance by the method disclosed in US7842793B2.

[0035] In some embodiments, the nanostructure(s) is protein based or peptide based where proteins or peptides can be natural or in silico generated or a combination, or comprises synthetic supramolecular polymers.

[0036] In some embodiments, the nanostructure(s) according to the invention are produced using nanolithography.

[0037] In some embodiments, the nanostructure(s) can be immobilized on a surface or can be in solution.

[0038] In some embodiments, the binding units’ corresponding binding partners are present on cells and said cells can be in solution or immobilized on a surface.

[0039] In some embodiments, the concentration of nanostructure(s) should be selected above overall binding avidity of the ensemble. To ensure the geometric communication the interaction between binding units and binding partners should be longer than thetimescale required for a biological response. As such the local concentration of the nanostructure(s) needs to surpass the concentration set by the dissociation constant.

[0040] In some embodiments, the nanostructure(s) is / are stabilized with coatings, preferably oligolysine (K10) PEG 1k / 5k.

[0041] As used herein, the term “identity” refers to the characteristic or property of being the same or identical. Specifically, it pertains to the similarity or equivalence of binding units in terms of their molecular composition, structure, or sequence (protein sequence, RNA sequence, nucleotide sequence, etc). It could be referred to as “molecular identity” or “sequence identity”.

[0042] When the description mentions "binding units of same or different identity," it means that the binding units can have similar or distinct structure or molecular compositions. They may be composed of the same type of molecules or different types, but they serve the common purpose of selectively interacting with their complementary partners on a biological interface.

[0043] For example, in the case of DNA origami, binding units with the same identity might consist of DNA strands with identical sequences, while binding units with different identities may have varying sequences. This diversity in binding unit identities provides versatility in the types of interactions that can be facilitated using the method outlined in the description.

[0044] The binding units can be covalently or non-covalently bound to the nanostructure. For example, the binding units can be indirectly or directly bound to the nanostructure via outwardly facing nucleic acid overhangs extending from the 3' and / or 5' ends of selected staple strands. In particular embodiments, the nucleic acid overhangs hybridize to a complementary target RNA, DNA or PNA sequence covalently linked to the binding units, by, for example, maleimide-thiol coupling.

[0045] When used in the context of a nucleic acid nanostructure object, “Staple strands” or “helper strands” refer to oligonucleotides that work as glue to hold the scaffold nucleic acid in its three-dimensional geometry. Additional nucleotides can be added to the staple strand at either 5' end or 3' end, and those are referred to as “staple overhangs”. Staple overhangs can be functionalized to have desired properties such as a specific sequence to hybridize to a target nucleic acid sequence, or a targeting element. Target nucleic acid sequences used to mask staple overhangs during the functionalization process are herein referred to as “guard strands”. In some instances, the staple overhang is biotinylated for capturing the DNA nanostructure on a streptavidin-coated bead. In someinstances, the staple overhang can be also modified with chemical moieties. Non-limiting examples include CLICK-chemistry groups (e.g., azide group, alkyne group, DIBO / DBCO), amine groups, and thiol groups. In some instances, some bases located inside the oligonucleotide can be modified using base analogs (e.g., 2-Aminopurine, Locked Nucleic Acids, such as those modified with an extra bridge connecting the 2' oxygen and 4' carbon) to serve as linker to attach functional moieties (e.g., lipids, proteins). Alternatively, DNA-binding proteins or guide RNAs can be used to attach secondary molecules to the DNA scaffold.

[0046] As used herein, "set angles" “set distances” refers to predetermined angles, distances or positions that are established for each binding units or between adjacent binding units on the nanostructure or between each binding units. These angles and distances (and / or positions) are deliberately designed and specified to create a particular geometric arrangement of binding units within the plane of the nanostructures. For example, if the binding units are arranged in a symmetrical pattern, the set angles and / or distances would dictate the precise orientation and spatial arrangement of each binding unit in relation to the others.

[0047] As used herein, "set positions” refers to predetermined positions that are established for each binding unit on the nanostructure. These positions are deliberately designed and specified to create a particular geometric arrangement of binding units within the plane of the nanostructures. For example, if the binding units are positioned on the edge platform, it could interact with a neighboring nanostructure and duplicate the imposed geometric arrangement. In some embodiments, this can be beneficial. In other embodiments, this can be detrimental. Inversely, if the binding units are positioned in the center, it can create negative space where no other imposed interaction can take place. In some embodiments, the resulting negative space can be beneficial. In other embodiments this can be detrimental.

[0048] In step b) of the method according to the invention, the nanostructure(s) is / are put in contact with the binding units’ corresponding binding partners to allow the organization of the binding partners in the imposed geometric arrangement. The first part of the step involves physically bringing the nanostructure(s) into contact with the binding partners. This contact can occur in a controlled environment, such as a laboratory setting, where precise conditions can be maintained, and for a duration that is adequate or appropriate for the binding units on the nanostructure to interact with their complementary partners on the biological interface. This duration is determined basedon factors such as the specific binding kinetics of the molecules involved, the nature of the biological interface, and the desired level of interaction. Essentially, it signifies the amount of time required to allow a meaningful and effective binding event to occur. This amount of time is usually correlated with the concentration of nanostructure(s) used. It ensures that the binding units have ample opportunity to engage with their respective partners in a manner that aligns with the objectives of the present invention. This duration can be comprised between days and minutes, preferably between 24h and 30 min, more preferably between 12h and 1h.

[0049] Step b) can be performed by short pulse and / or long exposure and / or by repeated dosing. In other terms, the nanostructure can be presented once, and / or through controlled release over a set time-range, and / or either of the previous presentation can be repeated to re-expose the binding target to the nanostructure.

[0050] "Corresponding binding partners" refers to the specific molecules or entities that have an affinity or compatibility with the binding units on the nanostructure. These are the complementary components that are designed to interact with the binding units in a specific manner.

[0051] Once in contact, the binding partners will interact with the binding units on the nanostructure. This interaction is guided by the specific geometric arrangement imposed by the binding units. As a result, the binding partners will organize themselves in a manner dictated by the defined spatial configuration.

[0052] The term “Imposed Geometric arrangement” refers to the predetermined arrangement and orientation of binding units on the nanostructure, as established in the earlier steps of the method. Said geometric arrangement can be defined as 2- dimensional as it refers to the arrangement of the binding units on the same plane of the nanostructure as viewed from above (see for instance Figure 1 to 8). The geometric arrangement serves as a template that guides the organization of the binding partners.

[0053] In the context of the invention, the particular geometric arrangement of the binding units on the nanostructures can create at least one specific pattern or shape on the surface (plane) of the nanostructure selected in the group consisting of points, lines, rays, line segments, triangles, which can be classified as equilateral, isosceles, scalene, right, acute, or obtuse, quadrilaterals that encompass squares, rectangles, parallelograms, rhombuses, trapezoids, and kites, other polygons including pentagons, hexagons, heptagons, octagons, nonagons, decagons, hendecagons, and dodecagons. Additionally, the patterns or shapes can be curved like ellipses, circles, semi-circles,arcs, sectors, segments, and annuluses, or more intricate patterns including ovals, hearts, infinity symbols («), spirals, sine and cosine waves, tessellations, lattices, grids, chevrons, checkerboards, herringbones, zigzags, honeycombs, and labyrinths, and combination therein.

[0054] See figure 3 for examples of patterns, see Figure 8 for an example of transmission Electron Microscopy imaging of geometric protein patterns, and figure 9 for an example of super resolution validation of geometric arrangement. See figure 11 for an example of the use of a geometric arrangement versus a random presentation of the same binding unit.

[0055] The particular geometric arrangement of the binding units on the nanostructures create specific patterns and shapes on the surface (plane) of the nanostructure that can be also be any symmetrical patterns or shapes comprising line symmetry, also known as reflectional symmetry, which occurs when one half of a shape is a mirror image of the other half along a defined axis; rotational symmetry which involves rotating a shape about its center point by a certain angle, less than 360 degrees, while still maintaining its original appearance; point symmetry which is present when a shape remains unchanged after a 180-degree rotation around its center point; translation symmetry which allows a shape to be moved along a straight line without altering its size, shape, or orientation; or glide reflection which combines translation with reflection, enabling a shape to be reflected and then slid along a line to a new position; Spiral symmetry which is a type of symmetry where a shape can be rotated around a central point and also scaled (enlarged or reduced) by a constant factor to map onto itself; gyration symmetry which is a combination of rotational and translational symmetry; bilateral symmetry which refers specifically to reflectional symmetry along a single line, meaning one half is a mirror image of the other; quadratic symmetry which involves a combination of rotational symmetry and reflectional symmetry; Fractal Symmetry which refers to the property where patterns within the shape repeat at different levels of magnification, resulting in a similar overall structure regardless of the level of detail observed.

[0056] In some embodiments, the distance between two adjacent binding units is comprised between 3 and 50 nm, or 15 nm to 50 nm, or 25 nm to 30 nm. A larger distance is not relevant as it foregoes a nanocluster, and a smaller distance is not relevant as it would be smaller than the size of the target binding partner.

[0057] In some specific embodiments, the distance between adjacent binding units is 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, 35, 40, 45, 50 nm.

[0058] In some specific embodiments, the angles between adjacent binding units is 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 45, 30,15, 10, 5° or combinations therein.

[0059] As used herein, “adjacent binding units” can refer to the binding unit or binding units in closest proximity to a reference binding unit. In particular embodiments, the adjacent binding unit must be on the same face of the nanostructure. In some embodiments, there will be two or more adjacent binding units to a single reference binding unit. Each adjacent binding unit can independently be another copy or copies of the reference binding unit or of a different identity. Thus, in some embodiments, all of the adjacent binding units are the same identity as the reference binding unit, all of the adjacent binding units are of a different identity from the reference binding unit, or the adjacent binding units are a combination of being of the same and different identities from the reference binding unit.

[0060] Binding units may be covalently or non-covalently attached to the nanostructure, and they may be cleavable by proteases or other enzymes or undergo triggered dissociation in response to environmental cues such as pH, etc. They and / or the nanoparticle may also be shielded from the immune system by encapsulating polymers or other materials for shielding and targeting purposes prior to antigen exposure at physiological sites of interest such as the injection site or within lymph nodes.

[0061] In some embodiments, binding units can be presented directly on the surface of the nanostructure without spacer.

[0062] In some other embodiments, binding units can be presented on the surface of the nanostructure with a rigid spacer, for instance dsDNA of 10-21 nt (example in figure 6 and 7). Short flexible section can be included to a maximum of 3.4nm extended length (e.g. 10nt). As shown in figure 10, flexible section can be at bottom, top or in the middle. In some embodiments, binding units can be equipped with a ssDNA strand with a complement sequence on the support, that is fully matching, or leaves space as single strand sections.

[0063] In some embodiments, the spacer can comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 single nucleotides or can be polymeric spacers of between 0 and 10 nm, preferably between 0 and 7 nm.

[0064] In some embodiments, polymeric spacers can be PEG, alkyl or a peptide (GGGS or other amino acid sequences). See figure 10.

[0065] In some embodiments, the spacer can be selected in the group consisting of: dsDNA of between 10 to 21 nt or less or equal than 21 nt, ssDNA of less or equal than 10nt, PEG of less or equal than 12 repeats, preferably less or equal than 10, more preferably less or equal than 6, Peptide GGGS (or variations therein, GSGG, GGSG...) of less or equal than 3 repeats, less or equal than 2, less or equal than 1 repeat of the 4 amino acid block, Peptide GG or GS repeats or small polar non charged amino acids, of less or equal than 6 repeats, less or equal than 4, less or equal than 2, Alkane (aliphatic) spacers, preferably C18, more preferably C12, even more preferably C6, Beta-alanine spacers of less or equal than 10 units, preferably less or equal than 8, more preferably less or equal than 6 units, Saccharides, for example hyaluronic acid, preferably of less or equal than 5 repeats, more preferably less or equal than 3 repeats.

[0066] These embodiments add flexibility in between the nanostructure and the binding unit. Flexibility diffuses the geometric arrangement area and creates surface overlap between binders and competition for the same binding site. The spacers should be rigid enough as to keep the imposed geometric arrangement. See figure 6 & figure 7 for examples of protein patterns with or without spacer.In some embodiments, flexibility of the binding unit is tolerated up to 20% of the nearest distance between adjacent binding units i.e the spatial tolerance of the binding unit position can deviate up to 20% of the distance to its nearest neighbor as long as the global pattern geometry is not affected.

[0067] In some embodiments, the binding units according to the invention are rigidly bound to the surface of the nanostructure.

[0068] In the context of the invention, the term "interaction" refers to the specific chemical or physical engagement between the binding units on the nanostructure and their complementary partners on the biological interface. This engagement involves the formation of specific bonds, forces, or interactions that allow the binding units to attach or associate with their respective counterparts for a long enough duration to impose the spatial geometry to the binding partners.

[0069] In some embodiments, the two or more copies of binding units according to the invention are of between two and five different identities, preferably between two and four different identities, more preferably between two and three different identities, evenmore preferably of two different identities, broadening therefore the applicability of the invention across a diverse range of molecular interactions.

[0070] See figure 1 for examples of binding units of the same identity or of different identities, with varying stoichiometry. The pattern is a hexagon for demonstration purposes not meant to be limiting.

[0071] See figure 2 for examples of binding units of different identities, with varying stoichiometry.

[0072] In some embodiments, the geometric arrangement is a symmetrical pattern. A symmetrical pattern enhances the predictability and reproducibility of interactions, further contributing to the precision of the method.

[0073] See figures 4 and 5 for examples of valency and symmetrical patterns of binding units of two different identities, in 1 :1 stoichiometry.

[0074] In some embodiments, the nanostructure is smaller than 100nmx100nm, preferably smaller than 60x60nm. The inventors observed that a larger nanostructure foregoes the communication with single clusters.

[0075] In some embodiments, the total number of copies of binding units is between 2 and 20, preferably between 4 and 15, more preferably between 6 and 12, even more preferably between 6 and 10. The number of binding units (valency) is sufficiently low - yet higher than 1 - to provide a distinguishable geometric arrangement. High valency does not allow for local communication and inherently results in general avidity enhancement of binding (classical multivalency).

[0076] In some embodiments, in step b) the nanostructure(s) is / are put in contact with the binding units’ corresponding binding partners present on a biomolecule, a cell membrane, a biological tissue, a living organism, or an organic or inorganic material.

[0077] As shown by the inventors the imposed geometric arrangement of binding partners present on a biological interface will influence the response induced by the binding unit. Said influence can be an activation, an inhibition, an upregulation, a downregulation, or a change in pathway due to geometric imposed new neighbors. The induced response can be selected from any cell reactions or the group consisting of Activation of certain pathway, Inhibition of certain pathway, Cell death, Adhesion, Mobility change, Morphological change, Phenotype change, Differentiation change, Maturation change, Proliferation change.

[0078] In another aspect of the invention, the invention relates to a nucleic acid nanostructure comprising(i) nucleic acids folded into a defined planar rigid nanostructure, and(ii) two or more copies of one or more binding units of the same or of different identity, Wherein the distance and the angles between adjacent binding units, the position of each binding unit on the surface of the nanostructure or a combination thereof improves or decreases and / or prolongs and / or shortens a response induced by the binding units relative to a control comprising the same binding units in an equal amount.

[0079] In some embodiments, the control differs from the nucleic acid nanostructure of the present invention regarding the distance and the angles between adjacent binding units and / or the position of each binding unit on the surface of the nanostructure.

[0080] Any of the nanostructures can further include one or more moieties incorporated in and / or linked to the nanostructure. Such moieties include, for example, adjuvants, targeting molecules, therapeutic agents, stabilizing agents, passivating agents, etc.

[0081] Cationic polymers and minor groove binders (as monomers, oligomers or polymers) may be used to coat the DNA nanoparticles for stabilization from endonuclease degradation. In particular, minor groove binders may act as tethers for covalent modifications of nucleic acids, for example to develop cross-linking strategies for DNA nanoparticles. These approaches can be combined, e.g. using brush or block copolymers, with PEGylation to further improve stabilization and passivation.

[0082] Nucleic acids for use in the described nanostructures can be synthetic or natural nucleic acids. In some embodiments, the nucleic acid sequences are not naturally occurring nucleic acid sequences. In some embodiments, the nucleic acid sequences are artificial or otherwise user defined nucleic acid sequences. Nucleic acid sequences that are artificial or otherwise user defined are typically non-naturally occurring nucleic acid sequences and can also be referred to as synthetic nucleic acid sequences.

[0083] In some embodiments, the nucleic acid nanostructures are not the genomic nucleic acid of a virus. In some embodiments, the nucleic acid nanostructures are viruslike particles.

[0084] Numerous other sources of nucleic acid samples are known or can be developed and any can be used with the described nanostructures, compositions and methods. In some embodiments, nucleic acids used in the described methods are naturally occurring nucleic acids. Examples of suitable nucleic acid samples for use within the described methods include DNA including genomic DNA samples, RNA samples, cDNA samples, nucleic acid libraries (including cDNA and genomic libraries), whole cell samples,environmental samples, culture samples, tissue samples, bodily fluids, and biopsy samples.

[0085] Nucleic acid fragments are segments of larger nucleic molecules. Nucleic acid fragments generally refer to nucleic acid molecules that have been cleaved. A nucleic acid sample that has been incubated with a nucleic acid cleaving reagent is referred to as a digested sample. A nucleic acid sample that has been digested using a restriction enzyme is referred to as a digested sample. In certain embodiments, the nucleic acid sample is a fragment or part of genomic DNA, such as human genomic DNA. Human genomic DNA is available from multiple commercial sources (e.g., Coriell #NA23248). Therefore, nucleic acid samples can be genomic DNA, such as human genomic DNA, or any digested or cleaved sample thereof. Generally, an amount of nucleic acids between 375 bp and 1,000,000 bp is used per nucleic acid nanostructure.

[0086] Although only a single nucleic acid strand is typically used as a scaffold sequence for folding the nanostructures, the reverse complement of the nucleic acid strand is used as an alternative for all applications.

[0087] M 13 is a common source of scaffold strand with native protein-coding sequence and approximately 7k bases. Sequence-controlled scaffold strands may also be produced using helper plasmids (Shepherd, et al., bioRxiv 521443 (2019), doi: https: / / doi.org / 10.1101 / 521443 and Praetorius et al., Nature, 552:84-87 (2017), Chasteen, et al., Nature, 34(21):e145 (2006)) or using a hybrid synthetic-enzymatic approach (Plesa, et al., Science, 359(6373): 343-347 (2018), DOI: 10.1126 / science.aao5167), or purely enzymatic approach (Veneziano, et al., Scientific Reports, 8, Article number: 6548 (2018)).

[0088] Systems and methods for the automated, step-wise design of a nucleic acid nanostructure having arbitrary spatial geometries are known in the art.

[0089] Scaffolded deoxyribonucleic acid (DNA) origami folds a long single-stranded DNA (ssDNA; “scaffold”) into a user-defined shape by slowly annealing the scaffold in the presence of shorter oligonucleotides (“staples”) containing segments or regions of complementary sequences to the scaffold that bring sequences that are far apart in sequence space to nearby locations in Euclidian space. These interactions and geometries are stabilized by specific Watson-Crick base pairing in the presence of salt that uses immobile Holliday junctions (“crossovers”) to constrain neighboring duplexes physically in space. Crossovers are generally engineered to occur between two parallel DNA duplexes at positions closest or nearest between the two or more helices of theDNA within a 1 D, 2D, or 3D structure. Scaffolded DNA origami was initiated by William Shih using a combination of parallel and anti-parallel crossovers (Shih, et al., Nature, 427(6975):618-21 (2004)) and subsequently Paul Rothemund using solely anti-parallel crossovers that has become the most ubiquitous form of scaffolded DNA origami (Rothemund, P W, Nature, 440, 297-302 (2006)), where Rothemund used M13 genomic ssDNA as the scaffold, and the technique has been further modified and generalized by numerous laboratories (Sharma, J et al., Science, 323, 112-116 (2009); Dietz, H. et al., Science, 325, 725-730 (2009); Douglas, S. M. et al., Nature, 459, 414-418. (2009);Brown, S et al., Nanoscale, 7, 16621-16624 (2015); Marchi, A. N. et al., Nano Lett, 14, 5740-5747 (2014)) using M13 or Phage lambda DNA.

[0090] Additional, top-down design of scaffolded DNA origami nanostructures have been demonstrated to automatically generate the scaffold routing and complementary ssDNA staple strands to self-assemble under appropriate folding conditions into user-defined geometries of 1 D, 2D, or 3D shapes (Veneziano, R et al., Science, 352, 1534 (2016); Benson, E et al., Nature, 523, 441-444 (2015); Douglas, S. M. et al., Nucleic Acids Res, 37, 5001-5006 (2009), Jun et al., ACS Nano, 2019, 10.1021 / acsnano.8b08671), and was the subject of work demonstrating generality of sequence design for scaffold DNA (see, for example, US20030215914A1 , US20050147962A1, WO2017089567A1 , W02017089570A1 , and CN106119269A).

[0091] One tile-based method allowed for generation of 2D wireframe objects (Yan, H et al., Science, 301 , 1882-1884 (2003)) that was subsequently implemented experimentally using M 13-based scaffolded DNA origami to a include diversity of 2D and closed 3D shapes (Zhang, et al., Nat Nanotechnol., 10(9):779-84 (2015)). This latter scaffolded DNA origami approach was subsequently generalized and fully automated for 3D shapes by Veneziano et al., (Veneziano, et al., Science, 352(6293): 1534 (2016)) for DX-based polyhedral origami and by Jun et al., ACS Nano, 2019, 10.1021 / acsnano.8b08671 for honeycomb and other polyhedral DNA origami.

[0092] Non-scaffolded DNA origami is an alternative approach that uses purely short strands of synthetic single-stranded DNA to self-assemble via thermally annealed folding large-scale arrays of structured DNA via a process known as ‘tile-based’ assembly (Yan, H et al., Science, 301 , 1882-1884 (2003); Winfree, E et al., Nature, 394, 539-544 (1998); Ke, Y et al., Science, 338, 1177-1183 (2012); Ke, Y et al., Nat Chem, 6, 994-1002 (2014)). In vivo production of top-down designed nanoparticles has long been one goal of the field, with recent promising successes in RNA and DNA (Elbaz, J et al., NatCommun, 7, 11179 (2016); Geary, C et al., Science, 345, 799-804 (2014); Nickels, P. C. et al., Small, 10, 1765-1769 (2014); Han, et al., Science, 358(6369) (2017)).

[0093] Historically, scaffolded DNA origami has largely relied on the natural M13 phage genomic single-stranded DNA as the scaffold (Rothemund, P W, Nature, 440, 297-302 (2006)). This is because it is natively single stranded and easy to produce in the bacteria E. coli, and therefore is available at low cost in large quantities. Efforts to increase production of M13 phage DNA have shown success, obtaining up to 410 mg of ssDNA from 1 liter of E. coli growth (Kick, B et al., Nano Lett, 15, 4672-4676 (2015)).

[0094] Additional composition and methods for making DNA origami structures are discussed in, for example, Dietz H et al (Dietz H et al., Science, 325, 725-730 (2009)), Liu et al (Liu et al., Angew. Chem. Int. Ed., 50, pp. 264-267 (2011)), Zhao et al (Zhao et al., Nano Lett., 11, pp. 2997-3002 (2011)), Woo et al (Woo et al., Nat. Chem. 3, pp. 620- 627 (2011)), Torrirg et al (Torring et al, Chem. Soc. Rev. 40, pp. 5636-5646 (2011), Shepherd, et al, (Shepherd, et al., bioRxiv 21443 (2019)), doi: https: / / doi.org / 10.1101 / 521443) and Praetorius et al (Praetorius et al., Nature, 552:84-87 (2017)).

[0095] In another aspect of the invention, the invention relates to an array of nucleic acid nanostructures according to the present invention.

[0096] Methods of selecting geometric arrangement of binding units on a nucleic acid nanostructure are also provided. The methods can include, for example, assaying the ability of two or more structurally different binding units-bound nucleic acid nanostructures to induce a response, wherein the two or more structurally different nucleic acid nanostructures differ by(i) the distance and the angles between adjacent binding units,(ii) the position of each binding unit on the surface of the nanostructure, or(iii) a combination thereof.

[0097] Said method of screening is used to select the most favorable imposed geometric arrangement for the most potent desired response induced by specific binding units.Description of the figures:

[0098] Figure 1: Examples of binding units positioned on a nanostructure and having either the same identity (2a), two different identities (2b), three different identities (2c) or four different identities (2d), with varying stoichiometry. One dot represents a singlebinding unit and one color represents one identity. The pattern is a hexagon for demonstration purposes not meant to be limiting.

[0099] Figure 2: Examples of binding units of different identities, with varying stoichiometry (1:1 , 1 :1 :1, 1 : 1 : 1 : 1 , 2:1, 2:1:1, 2: 1 :1 :1). One dot represents a single binding unit and one color represents one identity.

[0100] Figure 3: Example of patterns. The distance between single dots is 3, 4, 5, 6, 7, 8, 9, 10 nm.

[0101] Figure 4: Examples of the valency of binding units of two different identities, with the same stoichiometry (1 :1) in symmetrical patterns. One dot represents a single binding unit and one color represents one identity.

[0102] Figure 5: Examples of patterns of binding units of two different identities, with the same stoichiometry (1:1) in symmetrical patterns. One dot represents a single binding unit and one color represents one identity.

[0103] Figure 6: Example of protein pattern with spacer.

[0104] Figure 7: Example of protein pattern without spacer: the binding units are directly integrated in the nanostructure.

[0105] Figure 8: Transmission Electron Microscopy imaging of geometric protein patterns.

[0106] Figure 9: Super resolution validation of geometric arrangement. LH= large hexagon, SH= small hexagon, Lin= linear pattern. Scale bar is 15 nm.

[0107] Figure 10: super-selectivity response curves upon addition of flexibility in the geometric arrangement. Addition of 0T, 1T, 2T, 3T, 5T, 10T single strand DNA sections introduces spatial flexibility in the geometric arrangement and the pattern loses its clear definition. Data shows the loss of selectivity when flexibility is too high. A small amount of flexibility <10nt can sometimes be tolerated.

[0108] Figure 11: Use of a geometric arrangement versus a random presentation of the same binding unit. The binding interaction follows a super-selective multivalency mechanism for the uniform pattern, but a classic multivalent binding for the random presentation of binding units. Left: binding behavior for a geometric arrangement versus no pattern, super-selective versus multivalent, respectively. Right: Selectivity under 1 means not super-selective.

[0109] Figure 12: Analysis of the cell response depending on the geometric arrangement of the binding units by confocal microscopy 63X (see example 1).

[0110] Figure 13: Nanoplatform with binding units presented as mono ligands (RGD and PHSRN) and as dual ligands with different spacings, while a bare disk without ligand was used as negative control (see example 2).

[0111] Figure 14: Representation of a nanoplatform with binding units presented as dual ligands (RGD and PHSRN) (see example 2).

[0112] Figure 15: Response showed by Immunofluorescence stainings and confocal fluorescence microscopy of imposed 4 heteroclusters versus imposed 4+4 sparse pattern. The increased spacing between X and Y changes the cellular surface organization and imposes a different reaction by the cell. We observe multidirectional spreading and cytoskeletal organization for the close XY pattern, but unidirectional elongation for the far XY pattern.

[0113] Figure 16: See Example 3. (A) DNA origami disc library design and characterization (Left) Each white dot represents the 36 possible positions on the top side of the DNA origami disc available for ligand functionalization. (Right) the light grey dots indicate the positions for pMHC and the dark grey dots for PD-L1 molecules. DNA origami discs were either non-functionalized (“empty”) or functionalized either with pMHC only or a combination of pMHC and PD-L1. The latter were arranged into clusters that are either closely spaced (“close”, d~14nm) or widely (“far”, d~28nm) spaced.. (B) Agarose gel analysis (2% agarose) of the DNA origami disc library either functionalized (+ Ligands) or non-functionalized (no Ligand) with pMHC and / or PD-L1 molecules showing properly folded and purified samples. Cy5 signal characterizes assembled DNA origami disc and SYBR Safe signal represents all DNA-based samples. A delayed sample migration indicates successful attachment of the ligands. (C) Immobilization of DNA origami discs on culture plates for T cell proliferation assays. Quantification of DNA origami disc immobilization level was performed by measuring Cy5 intensity levels in each well. As a control, wells with 1x PBS were used. A similar plate coating level was observed across all samples.

[0114] Figure 17: See example 3. (A) (Left) Schematic illustrating MHC-I / PD-L1 spacing on B16F10 versus cDC1 cells, with the greyed out arrow above the ~30nm spacing indicating the hypothesis that far spacing will prevent PD-L1 mediated immunosuppression. (Middle) DNA origami disc nanoparticle-based approach to test whether close versus far spacing between peptide-MHC-l (pMHC-l) and PD-L1 promote T cell inhibition versus activation, respectively. pMHC-l and / or PD-L1 are adhered to the DNA disc via covalently attached DNA “handles” complementary to exposedoligonucleotide sequences on the disc. OT-I proliferation was measured after culture on plates coated with the relevant nanoparticle as a measure of T cell inhibition versus activation. (Right) Schematic illustration of the organisation of PD-L1 (dark grey) and OVA peptide-MHC-l complexes (pMHC; light grey) on the “Close” and “Far” nanoparticles. DNA origami structures without any attached protein (“Empty”) or with pMHC alone (“pMHC”) were included as negative and positive controls, respectively. (B) Relative OT-I cell count in each culture condition after three days of culture. Pooled data from three independent experiments are shown. Data was tested for statistical significance via a bootstrap ratio test. *** p < 0.001.

[0115] Figure 18: See Example 4. (A) design of flexible binding unit presentation, double black vertical lines represent double stand rigid DNA binding of the (round) binding unit to the DNA origami surface (grey rectangle). To the right, the binding unit is spaced by more and more ssDNA flexible sections (dotted lines, flexible spacer) representing 5T units or 10T units. (B) Design of rigid 6-valent hexagon (6HEX_0T) as geometric control, and increasing flexible 5T and 10T, similar for Linear geometry. (C) Flow cytometry results of early and late activation markers of primary T cells upon interaction with the rigid and flexible patterns as in Example 3. A remarkable strong negative impact of flexibility is observed.

[0116] Figure 19: See Example 5. (A) MHC-I patterns using 6 proteins through nanocontrolled display of these proteins on a previously validated, immobilized DNA origami platform (B) primary T cell response for early activation after 24h of the patterns of Fig. 19 (A). (C) Use of parallel patterns for co-presentation with PD-L1 to investigate how narrow the distance tolerance for T cell proliferation versus inhibition can be controlled. MHC-1 and PD-L1 were placed in parallel patterns with spacing 7.5 nm, 15 nm. 22.5 nm and 30 nm. (D) Measure of the expansion of primary T cells and the distance tolerance was found to be remarkably sharp. 15 nm spacing strongly inhibits proliferation, yet 22.5 nm strongly activates proliferation.Examples:

[0117] Example 1 : Example of cell communication through imposed spatial positioning

[0118] Herein is an example of cell communication through imposed spatial positioning of binding units at the cell interface, using the same binding unit but in different number and geometric arrangement and spacing. The geometry imposes a spatial organization on the cell membrane and the impact on cellular cytoskeleton organization is visualized.

[0119] The Pattern is designed on a DNA origami nanostructure, with cell adhesion peptides on the points. Either the same or different (below). The cells are allowed sufficient time to interact and respond.

[0120] Synthesis of particles:_Multivalent, Rigid DNA Scaffold were prepared as detailed a publication by Eklund et al. (ACS Nano 2021 , 15, 11, 17668-17677) Ligand functionalised ssDNA antihandle were annealed in 3X molar excess per handle to the Multivalent, Rigid Scaffold was performed in a Thermal Cycler with the following program : Samples were heated to 37°C for 2h, cooled to 28°C over 12h and stored at 4°C. 5pL, 10nM samples were loaded on 1% agarose gels with 1kb Plus ladder as reference. Peptide antihandle annealing was tested with further incubation with 3X molar excess of Cy5 conjugated antihandles. Gels to ensure folding and functionalisation were run for 90 mins at 70V.

[0121] Multivalent Scaffold surface immobilisation: Streptavidin was diluted to a concentration of 300nM in Mil liQ, coated on wells and stored at 4°C overnight. Wells were washed with PBS and blocked with 3% BSA for 30 minutes at 37°C prior to Multivalent Scaffold immobilisation. Multivalent Scaffolds, 4nM were immobilised for 30 mins at RT in EGM2- / Mg-Na / BSA (media without FBS supplemented with 18mM MgCI2, 5mM NaCI and 3% BSA). Wells were washed with EGM2- / Mg-Na / BSA prior to cell seeding.

[0122] Analysis of the cell response:_Cells were seeded for at least 24h at 37°C, 5% CO2, 95% relative humidity. Cells were serum starved overnight and incubated for 1h in full media prior to experiments. Cells were trypsinised, resuspended in EGM2- / Mg- Na / BSA, seeded in experimental wells and incubated at 37°C, 5% CO2, 95% relative humidity. After 1 h, the wells were washed with EGM2- / Mg-Na / BSA and incubated for a further 1h30. The following steps were conducted at RT. Cells were fixed in 2%PFA and 1X cytoskeleton buffer (1M NaCI, 0.1M PIPES, 30mM MgCI2, 10mM EGTA, 10mM sucrose) for 15min then washed with Buffer (18mM MgCh, 5mM NaCI, 5mM TRIS, 1mM EDTA) and imaged in Imaging Buffer (In PBS 1X : 0.5M NaCI, 18mM MgCh, Trolox 1X, PCA 1X, PCD 1X)

[0123] Stock solutions for the imaging buffers were prepared as follows: (i) Trolox 100X (100mg Trolox, 430pL 100% methanol, 345pL 1M NaOH, 3.2mL MilliQ) (ii) PCA 40X (154mg PCA in total 10mL MilliQ after pH 9.0 adjustment with NaOH) (iii) PCD 100X (9.3mg PCD in 13.3mL PCD buffer) (iv) PCD buffer (100mM TRIS-HCI pH8, 50mM KCI, 1mM EDTA, 50% glycerol)

[0124] Confocal microscopy 63X images of the relevant intracellular targets were acquired.

[0125] See figure 12: In white we see the intracellular organization of the cytoskeleton, which is either densely clustered in the center (top row) or more elongated and peripheral (bottom row).

[0126] Conclusion: Geometric arrangement more than valency (up to 6 is still low- valency regime) defines the cellular response. Imposing a large spacing prevents nanocluster formation. Imposing a close spacing imposes cellular clustering of membrane proteins, and triggers opposing cellular mechanisms.

[0127] Example 2: Example of cell communication through imposed spatial positioning of binding units at the biomembrane, using 2 different binding units in different spatial arrangements.

[0128] Annealing and purification of peptide-containing DNA disks: To synthesize the peptide-containing disks, reaction were performed using 10-30 nM of the bare disk and mixed with a 5-fold excess of peptide anti-handle per handle ( / .e. 20-fold excess in total) in folding buffer. The mixture was incubated at 37 °C for 1 h. Correct attachment of the peptide ligands to the disks was analyzed using 2 w / v% agarose gels. (70 V, 3 h). To remove excess peptide anti-handles, Amicon 30k MWCO filters were used. To prevent sticking of the DNA disks to the membrane and increase final yield, the filters were treated with Pluronic acid the day before and incubated at 4 °C overnight. The next day, Pluronic acid was removed, and the membranes were washed with MQ water by centrifuging at 16.000 g for 5 min and refilling it with MQ water (5x). The last cycle, the membranes were washed with folding buffer. Thereafter, the disks were added to the membrane and topped off with folding buffer. The samples were centrifuged at 5.000 g for 8 min. supernatant was discarded and the membranes were filled again with folding buffer, followed by similar centrifugation step. To recover the pure disks, the membrane was inverted in the tubes and subjected to a centrifugation step at 1.000 g for 2 min. The final yields were measured using nanodrop (260 nm).

[0129] See figure 13: Binding units are presented as mono ligands (RGD and PHSRN) and as dual ligands with different spacings, while a bare disk without ligand was used as negative control.

[0130] Surface immobilization of DNA disks onto SA coated surface: See figure 14. A stock of sterile SA was prepared at 1 mg / mL in PBS, which was diluted to 300 nM SA (fully saturated) in PBS. 10 pL of the 300 nM SA solution was pipetted into I bidi p-angiogenesis plates and the plate was incubated overnight at 4 °C. The next day, the solution was removed. Prior to adding the disks, disks were mixed with K10-PEG5k (dissolved in magnesium supplemented folding buffer, with an N:P ratio of 1 :1 , i.e. overall neutral) for 30 min at RT to make the disks inert. This step was executed to ensure the stiffness of the glass does not overrule the bioactive information that was offered to the cells. The plate was then incubated with the PEGylated, biotinylated disks in folding buffer (10 pL / well) using a concentration of 1 nM disks (fully saturated) for 1 h at RT. The plate was washed twice, before performing cellular experiments.

[0131] The cell was brought in contact for a duration sufficient to adhere and organize its internal cytoskeleton (stained in light grey) as well as its overall shape, changing from spread and round to long and elongated, depending on the pattern presented.

[0132] Immunofluorescence stainings and confocal fluorescence microscopy: cells were seeded on top of the pure DNA coatings and cultured for 1 day at 37 °C with 5% CO2. Next, the samples were washed with PBS, followed by fixation for 10 min with 3.7 v / v% formaldehyde in PBS and washing with PBS twice afterwards. The cells samples were then stained with phalloidin to visualize F-actin for 1 h at room temperature in the dark. Finally, the samples were washed with folding buffer. 15 pL folding buffer was left in the wells for the imaging. Immediately thereafter, the samples were imaged (and only mounted if necessary) on an inverted confocal microscope.

[0133] See figure 15 that shows the response to imposed 4 heteroclusters versus imposed 4+4 sparse pattern. The increased spacing between X and Y changes the cellular surface organization and imposes a different reaction by the cell. We observe multidirectional spreading and cytoskeletal organization for the closeXY pattern, but unidirectional elongation for the far XY pattern.

[0134] Surprisingly a minimal difference of 7 nm in the pattern is enough to induce a divergent cell response.

[0135] Example 3: Example of the method according to the invention with cells in solution

[0136] In this experiment the inventors elucidated whether the CD80-enforced nanoscale differences in MHC-I and PD-L1 pattern on the cell surface has functional consequences for T cell activation. To explore the biological impact of protein spacing and patterning, the inventors retro-engineered the identified MHC-I / PD-L1 consensus distances observed in cDC1 versus B16-F10 cells through nano-controlled display of these proteins on a previously validated, immobilised DNA origami platform (figure 16), and interrogated theprimary T cell response. After three days of co-culture, the close cluster pattern - matching the immuno-suppressive cancer-cell state - succesfully blocked T cell proliferation. Nonclustered MHC-I / PD-L1 failed to prevent T cell expansion, despite there only being a ~15nm difference in spacing between the two configurations. Taken together, the data show a clear functional relevance of the observed interaction patterns, underlining the profound regulatory effect of nanometer-scale pattern differences.

[0137] Preparation of the spatially organized materials. Example for two different binding targets.

[0138] DNA origami disc library design and characterization. See figure 16 (A) (Left) Each white dot represents the 36 possible positions on the top side of the DNA origami disc available for ligand functionalization. (Right) the dots indicate the positions for pMHC and the PD-L1 molecules. DNA origami discs were either non-functionalized (“empty”) or functionalized either with pMHC only or a combination of pMHC and PD-L1. The latter were arranged into clusters that are either closely spaced (“close”, d~14nm) or widely (“far”, d~28nm) spaced. (B) Agarose gel analysis (2% agarose) of the DNA origami disc library either functionalized (+ Ligands) or non-functionalized (no Ligand) with pMHC and / or PD-L1 molecules showing properly folded and purified samples. Cy5 signal characterizes assembled DNA origami disc and SYBR Safe signal represents all DNA- based samples. A delayed sample migration indicates successful attachment of the ligands. (C) Immobilization of DNA origami discs on culture plates for T cell proliferation assays. Quantification of DNA origami disc immobilization level was performed by measuring Cy5 intensity levels in each well. As a control, wells with 1x PBS were used. A similar plate coating level was observed across all samples.

[0139] Once the spatially organized materials prepared, the cell culture experiment was performed: In this experiment, the inventors looked at the proliferation of primary T cells, isolated from mice.

[0140] T cell isolation. Mouse splenocytes and lymphocytes were extracted by mashing spleen and axillary and inguinal LNs through a 40 pm cell strainer. Primary OT-I CD8+T cells were subsequently isolated using the EasySep mouse CD8+T cell isolation kit, following manufacturer’s instructions. Until further use, pure CD8+T cells were kept in T cell medium (RPMI-GlutaMAX™ supplemented with 10% heat-inactivated FBS, 50 U / mL penicillin, 50 pg / mL streptomycin, 10 mM HEPES, and 50 pM p-mercaptoethanol) at 4 °C. The purity of isolated CD8+T cells was assessed using the LSRII SORP flow cytometer (BD Biosciences) by staining CD8+T cells with FITC anti-CD3, PE-Cy5.5 anti-CD8a, andFixable Viability Dye eFluor™ 780. The purity was systematically higher than 90%. Data processing was performed using FACS Diva (BD Biosciences) and FlowJo (Tree Star).

[0141] In vitro T cell proliferation assay. Prior to CD8+T cells seeding, high-binding 96- well plates were pre-treated with streptavidin, incubated with a blocking solution (3% BSA in PBS) and coated with the corresponding biotinylated DNA origami disc samples. DNA disc immobilization level was quantified using the Cy5 intensities measured with Cytation TM5 plate reader (BioTek Instruments). CD8+T cells were then cultured on DNA origami disc plates in T cell medium supplemented with 0.5 pg / mL anti-CD28, 10 ng / mL IL-2, 5 ng / mL IL-7, 50 pg / mL primocin and 10 mM MgCh, and maintained at 37 °C and 5% CO2. T cells were cultured for a total of 72h and every 24h cells were transferred on freshly prepared DNA origami disc plates. On day 3, CD8+T cells were collected and stained with PE anti-CD3 and Live / dead fixable blue dead cell stain. Cell count was assessed by quantifying the cell number on day 3 using Precision Counting Beads and the Fortessa flow cytometer (BD Biosciences) and tested for statistical significance via a bootstrap ratio test(58, 59). Each independent experiment was performed using primary T cells isolated from a different OT-I mouse. See Figure 17.

[0142] Conclusion: Using a double geometry of symmetric linear pattern in low valency (8 units), surprisingly, this two-lines pattern with a minimal distance of 15nm between the two lines allowed to inverse T cell reaction from strong proliferation to strong inhibition.

[0143] Example 4: example of the method according to the invention showing the critical effect of flexibility

[0144] In this experiment the inventors elucidated the remarkable effect of local flexibility in changing the cellular reaction toward binding molecules presented in geometric patterns. MHC-I patterns on the cell surface have functional consequences for T cell activation. To explore the biological impact of binder flexibility, the inventors engineered MHC-I patterns based on the methods used in Example 3. Hexagonal and linear pattern were chosen for this study, and the MHC-I proteins were presented with increasing local flexibility, by adding 5T or 10T unpaired ssDNA section in the handle docking strand (figure 18a, b). The inventors then interrogated the primary T cell response after24h, 48h, and 72h to study the biological early and late activation pathways by flow cytometry via the markers CD69, CD25 and CD137 respectively (figure 18c). Surprisingly, strong decrease of activation of all markers was observed with increasing flexibility as well as a pattern dependent effect for late activation. Taken together, the data show a clear functional relevance of the observed changes in flexibility patterns, underlining the 1profound regulatory effect not only of geometric pattern and valency, but also flexibility differences.

[0145] Example 5: Example of the method according to the invention with cells in solution with different geometry and distance spacing

[0146] In this experiment the inventors elucidated whether geometric patterns of MHC-I alone has functional consequences for T cell activation. The preparation of the spatially organized materials is the same as in example 3.

[0147] To explore the biological impact of protein spacing and patterning, the inventors engineered MHC-I patterns using 6 proteins through nano-controlled display of these proteins on a previously validated, immobilised DNA origami platform (figure 19 A, B), and interrogated the primary T cell response for early activation after 24h. Data shows that a clear and significant pattern effect is present and that hexagonal organization is causing the strongest early activation of T cells followed by parallel pattern. Next, the parallel pattern was used for co-presentation with PD-L1 to investigate how narrow the distance tolerance for T cell proliferation versus inhibition could be controlled. MHC-1 and PD-L1 were placed in parallel patterns with spacing 7.5 nm, 15 nm. 22.5 nm and 30 nm (figure 19 C). After three days of co-culture, the expansion of primary T cells was measured and the distance tolerance was found to be remarkably sharp (figure 19 D) 15 nm spacing strongly inhibit proliferation, yet 22.5 nm strongly activates proliferation. Taken together, the data show a clear functional relevance of the observed interaction patterns using both geometry and the distance between 2 binding unit types, underlining the profound regulatory effect of nanometer-scale distance differences and geometric rigid patterns.

Claims

CLAIMS1. Method to impose a geometric arrangement to binding partners present on a biological interface comprising the steps of: a) providing planar rigid nanostructure(s) comprising two or more copies of one or more binding units of the same or of different identity bound to the surface of the nanostructure;Wherein said binding units comprise set angles and set distance(s) between adjacent binding units and / or a set position on the surface of the nanostructure; b) putting the nanostructure(s) in contact with the binding units’ corresponding binding partners to allow the organization of the binding partners in the imposed geometric arrangement.

2. Method according to claim 1 , wherein said two or more copies are of two or more binding units of between two and five different identities, preferably between two and four different identities, more preferably between two and three different identities, even more preferably of two different identities.

3. Method according to any one of claims 1 to 2, wherein the particular geometric arrangement of the binding units on the nanostructure(s) create at least one specific patterns and shapes on the surface of the nanostructure selected in the group consisting of points, lines, rays, line segments, triangles, which can be classified as equilateral, isosceles, scalene, right, acute, or obtuse, quadrilaterals that encompass squares, rectangles, parallelograms, rhombuses, trapezoids, and kites, other polygons including pentagons, hexagons, heptagons, octagons, nonagons, decagons, hendecagons, and dodecagons, curved patterns or shapes such as ellipses, circles, semi-circles, arcs, sectors, segments, and annuluses, or more intricate patterns including ovals, hearts, infinity symbols («), spirals, sine and cosine waves, tessellations, lattices, grids, chevrons, checkerboards, herringbones, zigzags, honeycombs, and labyrinths, and combination therein.

4. Method according to any one of claims 1 to 3, wherein the particular geometric arrangement of the binding units on the nanostructure(s) create at least one symmetrical pattern, selected in the group consisting of line Symmetry (Reflectional Symmetry), rotational Symmetry, point Symmetry, translation Symmetry (slide Symmetry), glide Reflection, spiral Symmetry, gyration Symmetry, bilateral Symmetry, quadratic Symmetry, fractal Symmetry, and combination therein.

5. Method according to any one of claims 1 to 4, wherein said two or more binding units are spaced apart with a distance of between 3 and 50 nm.

6. Method according to any one of claims 1 to 5, wherein the nanostructure is smaller than 100nm x 100nm.

7. Method according to any one of claims 1 to 6, wherein the total number of binding units is between 2 and 20, preferably between 4 and 16, even more preferably between 6 and 10.

8. Method according to any one of claims 1 to 7, wherein said nanostructure is produced using nanolithography or DNA origami and / or is a nano-disk and / or is a nucleic acid nanostructure.

9. Method according to any one of claims 1 to 8, wherein in step b) the nanostructure(s) is / are put in contact with the binding units’ corresponding binding partners present on abiomolecule, a cell membrane, a biological tissue, or a living organism or an organic or inorganic material.

10. Method according to any of claims 1 to 9, wherein the binding units are presented on the surface of the nanostructure without a spacer or with a rigid spacer or where the flexibility of the spacer does not alter the geometric position and pattern, e.g. the spatial tolerance of the flexible spacer is significantly shorter than the size of the binding unit.

11. A nucleic acid nanostructure comprising(i) nucleic acids folded into a defined planar rigid nanostructure, and(ii) two or more copies of one or more binding units of the same or of different identity, Wherein the distance and the angles between adjacent binding units or the position of each binding unit on the surface of the nanostructure improves or decreases a response induced by the binding units relative to a control comprising the same binding units in an equal amount.

12. A nucleic acid nanostructure according to claim 11, wherein the control differs from the nucleic acid nanostructure of claim 11 regarding the distance and the angles between adjacent binding units, the position of each binding unit on the surface of the nanostructure, the particular geometric arrangement of the binding units on the nanostructure(s) creating at least one specific patterns and shapes on the surface of the nanostructure, or a combination thereof.

13. The nanostructure according to any one of claims of claims 11 or 12, wherein the total number of copies of binding units is between 2 and 20, preferably between 4 and 15, more preferably between 6 and 12, even more preferably between 6 and 10.

14. The nanostructure according to any one of claims 10 to 13, wherein the distance between two adjacent binding units is comprised between 3 and 50 nm, preferably 15 nm to 50 nm, more preferably 25 nm to 30 nm.

15. Method of selecting a geometric arrangement of binding units on a nucleic acid nanostructure according to claims 11 to 14 comprising assaying the ability of two or more structurally different binding units-bound nucleic acid nanostructures to induce a response, wherein the two or more structurally different nucleic acid nanostructures differ by:(i) the distance and the angles between adjacent binding units,(ii) the position of each binding unit on the surface of the nanostructure, or(iii) a combination thereof.

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