[TITLE]

A NUCLEIC ACID ORIGAMI MODULAR DEVICE

[TECHNICAL FIELD]

[0001] The disclosure relates to a nucleic acid origami modular device which can be used for exerting high throughput mechanical forces and constraints on surfaces, such as biological surfaces, and which uses DNA origami components configured to cooperate together to exert such forces and constraints.

[TECHNICAL BACKGROUND]

[0002] Mechanical forces at the nanoscale are essential for numerous biological processes. To directly detect these forces, cells use molecular mechanosensors, such as the cytoskeleton, molecular motor proteins, and cell surface receptors to translate piconewton (pN) scale mechanical forces into biochemical signals (Ma et aL, Small, 15, 1900961., 2019). Investigating these molecular systems has been facilitated by the development of techniques that interface with molecules, such as atomic force microscopy (AFM), magnetic, and optical tweezers (Nathwani et al. Biophys J 1 15, 2279-2285, 2018).

[0003] Significant discoveries have been made using these techniques. However, these tools possess some remaining drawbacks which limit their usage. Indeed, these tools present the disadvantages to be expensive and laborious. In particular, these techniques may be used only in a low throughput with low molecular specificity. AFM methods have difficulties to operate at a low pN range of force suitable for a majority of biological effects. Also, optical tweezers have proven useful for mechanochemical sensing. However, the throughput of the sensing is low since each time only one template can be investigated. In addition, they require connection of the biomolecule to long tethers or to operate exclusively on a supporting surface.

[0004] Nucleic acid nanotechnology is one promising approach, able to interface with living cells, as well as to measure and exert pN scale forces. For example, synthetic DNA has been at the forefront of probes designed to interrogate mechanosensitive cell surface receptors using single-molecule fluorescence. Moreover, it allows the fabrication of sophisticated nucleic acid origami devices with complex geometries, programmable mechanical properties, and precise positioning of molecular functionalities (Douglas, et al. Nature 459, 414-418 (2009); Dutta et al. Nano letters, 18, 4803-4811 (2018)). [0005] DNA origami methods have previously been applied to decrease experimental noise in optical tweezers (Kilchherr, et al. Science, 353, 6304 (2016)) and introduce massive parallelization and high data throughput in single-molecule fluorescence (Nickels et al. Science, 354, 305-307 (2016)). DNA origami are now being encoded for creating machine-like nanostructures that can be mechanically controlled through the hybridization of oligonucleotides, thermal fluctuations, and electrical fields (Marras et aL, PNAS, 112(3):713-8. (2015); Ramezani et aL, Nature Reviews Genetics (1 ):5-26 (2020) ; Funke et aL, Science Advances. 11 , e1600974 (2016); Kopperger et aL, Science, 359, 296- 301 (2018)).

[0006] There remains a need for tools for measuring or exerting pN scale forces on surfaces of interest, for example biological surfaces, such as cell surface.

[0007] There is a need for tools for measuring or exerting pN scale force with a high- throughput.

[0008] There is a need for tools able to exert a force on a surface of interest, independently of the attachment of the tool and/or the surface on a secondary support, such as an arm or a cell culture plate.

[0009] There is a need for tools able to specifically attach to a surface of interest.

[0010] There is also a need for tools able to operate on surface of cells in suspension.

[0011] There is a need for tools able to operate at specific location of a surface of interest.

[0012] There is a need for tools able to be controlled at nanoscale.

[0013] There is a need for tools able to be remotely controlled.

[0014] There is a need for tools which exert mechanical forces at nanoscale.

[0015] There is a need for tools which exert mechanical constraints at nanoscale.

[0016] There is also a need for cost-effective tools.

[0017] There is a need for cost-effective tools able to operate at nanoscales.

[0018] There is a need for cost-effective tools able to operate at a high-throughput debit.

[0019] The present disclosure has for purpose to satisfy all or part of those needs. [SUMMARY]

[0020] According to one of its objects, the disclosure relates to a nucleic acid origami modular device comprising a first component and a second component, each of the first and second component comprising a nucleic acid scaffold strand and a plurality of nucleic acid staple strands, the first component and second component being attached together by hybridization of at least two staple strands, the first component being at least one nucleic acid origami support and the second component being a nucleic acid origami module, wherein the nucleic acid origami support comprises at least one anchor element for attaching the nucleic acid origami modular device to a surface of interest, and the nucleic acid origami module being configured for interacting with said surface of interest.

[0021] According to another of its objects, the present disclosure relates to a nucleic acid origami modular device comprising a first component and a second component, each of the first and second component comprising a nucleic acid scaffold strand and a plurality of nucleic acid staple strands, the first component and second component being attached together by hybridization of at least two staple strands, the first component being at least one nucleic acid origami support and the second component being a nucleic acid origami module, wherein the nucleic acid origami support comprises at least one anchor element for attaching the nucleic acid origami modular device to a surface of interest, and the nucleic acid origami module being configured for interacting with said surface of interest wherein the first and the second components are arranged to cooperate together for exerting a mechanical force on the surface of interest.

[0022] The mechanical force exerted on the surface of interest may induce a mechanical constraint.

[0023] The nucleic acid origami module may be an actuator.

[0024] As shown in the Examples section, the inventors have reported the manufacture of a nucleic acid origami modular device able to exert linear mechanical constraints on cell surface or on specific, individual cell membrane protein.

[0025] As shown in the Examples, the nucleic acid origami modular device of the disclosure is a tunable, adjustable nanodevice able to interface directly with molecular systems and cells to elicit biochemical effects via mechanical tension. [0026] Advantageously, the nucleic acid origami modular device of the disclosure allows for expanding the palette of techniques to investigate mechanical-chemical communication of cells.

[0027] Furthermore, the components of the nucleic acid origami modular device are simple to assemble into a complete actuator, robust enough to be used in a variety of systems and able to adjust the applied distance and tension.

[0028] Functionalization of the anchor elements of the nucleic acid origami supports with molecules, such as cholesterol, may be a general method to attach the device to a membrane bilayer. Advantageously, it is possible to functionalize the anchor element with other ligands, such as antibodies or nanobodies, to target specific cell types.

[0029] Furthermore, functionalization of the nucleic acid module with specific binding moiety in a controlled stoichiometry and vicinity allows for providing opportunities for interactions with different membrane proteins and for multivalent interactions.

[0030] The nucleic acid origami modular device manufacturing method allows cost- effectively producing billions of functionalized nano-devices able to work in parallel to mechanically activate membrane proteins, and to reach high-throughput data acquisition.

[0031] The nucleic acid origami modular devices may be produced in thousands or millions or billions of units at low cost, and can be used for high-throughput measures.

[0032] The use of thousands or millions or billions of nucleic acid origami modular devices of the disclosure allow to exert or measure pN or hundreds of pN forces at a high- throughput debit. For example, as disclosed hereafter, it is possible to associate a fluorescent or chemiluminescent signal to a mechanical constraint exerted by a nucleic acid origami modular device of the disclosure. The possibility to use thousands of devices at a surface of a cell allows to amplify the fluorescent or chemiluminescent signal emitted and to obtain a stronger and more reliable measure.

[0033] The first and the second components may be arranged to cooperate together for exerting a force, in particular a mechanical force, on the surface of interest. The exerted force, in particular the exerted mechanical force, may induce a mechanical constraint or stress.

[0034] In some embodiments, at least two nucleic acid origami supports may be attached to the nucleic acid origami module.

[0035] The nucleic acid origami support may comprise a head for attaching the nucleic acid origami support to the nucleic acid origami module, and at least two extended members projecting from said head, said each extended members being connected by one end to said head and by the other end to at least one base, said base comprising said at least one anchor element.

[0036] A base may comprise from one to twelve anchor elements. The anchor element may be a nucleic acid staple strand. The nucleic acid staple strand may be functionalized with at least one moiety suitable to adhere to or insert in said surface of interest. An anchor element may comprise at least one ligand-conjugated oligonucleotide. A ligand-conjugated oligonucleotide may be a cholesterol-conjugated oligonucleotide.

[0037] The extended member may be further connected to the base by at least one nucleic acid connector strand.

[0038] The nucleic acid origami module may comprise a sleeve and an extended element, said sleeve and said extended element each comprising a longitudinal axe, said extended element being disposed concentrically within said sleeve, with the longitudinal axe of said extended element being parallel to the longitudinal axe of the sleeve, and said extended element and said sleeve being movable one relative to the other.

[0039] The extended element may comprise a first and a second end, the first and second end being each connected with the sleeve with at least one nucleic acid connector strand.

[0040] One end of the extended element may comprise a head. The head of the extended element may comprise transversal dimension, perpendicular to the longitudinal axe of the extended element, which is larger than the transversal dimension of the sleeve, perpendicular to its longitudinal axe. The head of the end of the extended element may prevent the sleeve to move beyond this end of the extended element.

[0041] The nucleic acid origami module and the nucleic acid origami support may be arranged together for having the longitudinal axe of the extended element the nucleic acid origami module perpendicular to the surface of interest, when the nucleic acid origami modular device is positioned at the surface of interest.

[0042] An end of the extended element may be proximal to the surface of interest and the other end may be distal from the surface of interest. The proximal end may be functionalized with at least one binding moiety able to bind a complementary moiety present at the surface of interest.

[0043] The binding moiety may be an RGD-containing peptide.

[0044] The distal end may comprise a head as above indicated. [0045] At least one nucleic acid connector strand may connect the head of the end of the extended element and the sleeve.

[0046] A portion of the least one nucleic acid connector strand connected to the sleeve and the end of the extended element distal from the surface of interest may be folded on said distal end.

[0047] A portion of the least one nucleic acid connector strand connected to the sleeve and the end of the extended element distal from the surface of interest may be folded on said head of said distal end.

[0048] A portion of the least one nucleic acid connector strand connected to the sleeve and the end of the extended element distal from the surface of interest may be folded in a form of a loop, preferably in a form of a plurality of continued loops.

[0049] A portion of the least one nucleic acid connector strand connected to the sleeve and the end of the extended element distal from the surface of interest may be an entropic spring. The entropic spring may exert a force moving back and forth the extended element within the sleeve in a direction parallel to the longitudinal axes of the extended element and of the sleeve.

[0050] According to another of its objects, the disclosure relates to a method for preparing a nucleic acid origami modular device as disclosed herein, the method comprising at least a step of mixing a first composition comprising nucleic acid origami supports as disclosed herein and a second composition comprising nucleic acid origami modules as disclosed herein.

[0051] According to another of its objects, the disclosure relates to a kit for preparing a nucleic acid origami modular device of the disclosure comprising a first container comprising a first composition comprising nucleic acid origami supports as disclosed herein and a second container comprising a second composition comprising nucleic acid origami modules as disclosed herein.

[0052] According to another of its objects, the disclosure relates to a use of a nucleic acid origami modular device according of the disclosure for exerting a force on the surface of interest, optionally said force inducing a mechanical constraint.

[0053] According to another of its objects, the disclosure relates to a method for exerting a force, optionally for inducing a mechanical constraint, on a surface of interest, the method comprising at least a step of contacting a nucleic acid origami modular device of the disclosure with said surface, in conditions suitable for the nucleic acid origami support to attach to said nucleic acid origami modular device to said surface of interest, and for the nucleic acid module to interact with said surface of interest, wherein said interaction exerts a force on said surface of interest.

[0054] The force, in particular the mechanical force, may induce a mechanical constraint.

[0055] A mechanical constraint may be a traction or a pression. A mechanical constraint may be a mechanical traction or a mechanical pression.

[0056] The method may further comprise a step of hybridizing a nucleic acid strand with one nucleic acid connector strand connecting an end of the extended element and the sleeve. The end may be the end distal from the surface.

[0057] According to one of its objects, the present disclosure relates to a surface of interest comprising attached to it a nucleic acid origami modular device according to the disclosure.

[DESCRIPTION OF THE FIGURES]

[0058] Figure 1 represents the design of a DNA origami modular device. Schematic illustration of the assembly strategy of the DNA origami modular device. Double-helical DNA domains are represented by cylinders and are packed on a honeycomb lattice. The DNA origami modular device comprises two origami in a 1 :2 trimer, a central DNA origami module, which correspond to an extended element having a head and a sleeve, and two DNA origami supports each correspond to a head, an extended member and a base. The DNA origami module has eight single-stranded DNA (represented by (i)) to anneal to the inner face of the two supports. To prevent toppling, two bases, which correspond to about 30nm six-helix bundles, project at 90° from the extended members of the supports and 45° away from each other to lay parallel to a surface and maximize area coverage to retain an upright position. The base and the extended member are reinforced with a transversal connector which is a dsDNA structure. Single-stranded DNA connectors (represented by (ii)) link the top and the bottom of the sleeve to the head and the end of the extended element of the DNA origami module, respectively. These single-stranded DNA connectors loops act as entropic springs with stiffness D NA and exert defined force which is mechanically translated through the rigid origami to the tip of the piston coupled to a molecular target with stiffness kprotein. The length of these connector loops can be adjusted by storing the excess scaffold in reservoir loops on the head of the piston (represented by (iii)). The end of the extended element positions up to three ligand moieties targeting specific molecule on a surface of interest (represented by (iv)). Extension of the DNA origami module results in equivalent and opposing compressive force through the DNA origami supports.

[0059] Figure 2 represents the assembly of a DNA origami modular device. (Figure 2A) The following samples were electrophoresed on a 1.0% agarose gel with 1 1 mM of MgCls ; (a) M (Molecular weight marker - 1000 bp), (b) DNA origami module monomer (Piston-cylinder monomer), (c) DNA origami support monomer (Landing Leg monomer), and DNA origami modules incubated with two-fold molar excess of DNA origami supports with a concentration gradient (d)1 1 mM, (e) 20 mM, (f) 25 mM, (g) 30mM, and (h) 35 mM of magnesium (MgCh). (Figure 2B) Schematic representations and reference-free class averages from single-particle TEM micrographs of individual components. (Figure 2C) Fully assembled DNA origami modular devices were visualized both laterally and from above.

[0060] Figure 3 represents a DNA origami modular device functionalized with cholesterol moieties. Each base of the DNA origami supports can be modified with eight cholesterol moieties, for a total of 32-modifications. Example TEM images of DNA origami modular device functionalized with cholesterol moieties adhering to small unilamellar vesicles through the bases of the supports. Distortions of liposomes is an effect of the process of sample preparation for negative stain TEM. White bars represent 50nm. All TEM analyses were conducted at least three times for each sample.

[0061] Figure 4 represents the autonomous single-stranded DNA origami modular device characterization. (Figure 4A) Fully assembled trimer with single-stranded 97nt connectors exists in an equilibrium state between about 5 nm to 25nm with corresponding reference-free class average calculated from single-particle TEM micrographs. Measured distance r, correlates directly to the distance d between the surface of interest and the end of the extended element of the DNA origami module. DNA origami modular device tuned with (Figure 4B) 97 nucleotides, (Figure 4C) 60 nucleotides, and (Figure 4D) 30 nucleotides connector strands and the corresponding distance d distributions and reference-free class averages. The length of the connectors is adjusted by storing the excess strands in reservoir loops on the head of the piston ((iii) in Figure 1 ).

[0062] Figure 5 represents the forces exerted by a single-stranded DNA origami modular device (Figure 5A) The forces exerted by a single strand connector on the head of the extended element of the module (n=1 ) (“ssDNA up”) and on the end of the extended element (“ssDNA dw”) 97-nt ssDNA molecule are plotted as a function of extension x defined as the distance between the end of the extended element of the module and the bottom of the sleeve. The force of the linear spring is plotted for the values of kprotein (kpr) = (0.1 , 0.5, 1 ) pN/nm. (Figure 5B) Total force as a function of x for the three values used for kpr. The corresponding values of the equilibrium state of x, x _eq , are shown as vertical gray dashed lines in both a) and b). (Figure 5C) The total force applied on the linear spring as a function of kpr, when n=6 molecules of 97 nucleotides are present at the head and at the end of the DNA origami module.

[0063] Figure 6 represents the autonomous DNA origami modular device activation of integrin signaling. (Figure 6A) The transmembrane receptor integrin exists as a compact ap heterodimer. Integrins transmit applied mechanical stresses, between 1 and 15 pN, and recruits additional proteins to assemble focal adhesions including Focal Adhesion Kinase (FAK), which becomes phosphorylated at residue Y397 after mechanical stimulation of integrin. Addition of two antibodies with donor, D, and acceptor, A, labels allow detection of phosphorylated FAK in a LRET assay. Both antibodies bind to phosphorylated FAK (Y397- P) eliciting a detectable high LRET signal, whereas only a single antibody binds in the absence of phosphorylation yielding a low LRET signal. (Figure 6B) MCF-7 cells in suspension were: (sample 1 ) untreated control, (sample 2) incubated with RGD-conjugated oligonucleotide, (sample 3) incubated with cRGD functionalized DNA origami module, (sample 4) incubated with non-functionalized DNA origami modular device, (sample 5) incubated with cRGD functionalized DNA origami modular device. Cells were then lysed and a FAK phosphorylation was applied. The background signal, Ro, of antibodies alone was subtracted from the signal of lysed cells in experimental and control conditions calculated from ratios of acceptor and donor fluorescence intensities, RAD. Results are the average of at least three independent experiments. Error bars represent the standard deviation, statistical significance was determined by one-way analysis of variance with comparison to the untreated control (***, P <0.001 ).

[0064] Figure 7 represents the remotely activated double-stranded DNA origami modular device characterization. Addition of extension oligonucleotides complementary to the strand connectors between the head of the module and the sleeve ratchets the module from the membrane of the surface of interest. Annealing of the oligonucleotides to these connector strands transitions them from single-stranded, with a persistence length of about 1 nm, to double-stranded, which is fifty times stiffer, at 50nm.

[0065] Figure 8 represents the activity of the remotely activated double stranded DNA origami modular device. (Figure 8A) A DNA origami modular device tuned with 30 nucleotides strand connector between the head of the extended element of the module and the sleeve with corresponding distance d measurements and reference-free class averages. (Figure 8B) A DNA origami modular device tuned with 60 nucleotides strand connector between the head of the extended element of the module and the sleeve with corresponding distance d measurements and reference-free class averages.(Figure 8C) A DNA origami modular device tuned with 97 nucleotides strand connector between the head of the extended element of the module and the sleeve with corresponding distance d measurements and reference-free class averages.

[0066] Figure 9. The mean distance extension of 30 nucleotides, 60 nucleotides, and 97 nucleotides strand connectors (black dots) overlaid on the WLC model of DNA origami module extension (line linking dots) enables the user to rationally define the strand connectors length for a required distance d. Error bars represent standard deviation.

[0067] Figure 10. A DNA origami modular device was folded with a DNA hairpin tethering the head of the extended element to the sleeve requiring F1/2 of approximately 20pN to unzip. This hairpin serves as a benchmark to cross-validate the force applied by annealing of the extension oligonucleotides estimated by coarse-grained molecular dynamics simulation.

[0068] Figure 11. The distance d of tethered DNA origami module with the hairpin were measured before (distance of 0 nm to 18 nm) and after (distance of 19 nm to 40 nm) incubation with extension oligonucleotides.

[0069] Figure 12 represents a demonstration of the activity of the remotely activated double stranded DNA origami modular device. BtuB is a mechanically gated 0-barrel channel occluded by a globular plug domain. The N-terminal 49 residues (dark grey part of the plug domain) are dislocated to form a channel and a N- terminal linker was engineered with a cysteine residue into the protein to serve as a linker for conjugation with a thiolated oligonucleotide. Addition of extension oligonucleotides retracts the extended element o the module and unfolds part of the plug domain to open a channel.

[0070] Figure 13 represents the remotely activation of the device (following figure 12). (Figure 13A) Selected traces and corresponding trace count histogram of BtuB-oligo reconstituted into planar lipid bilayers with DNA origami modular devices after addition of 400 nM extension oligonucleotides. Closed channels (light grey) transitioned to an open state (dark grey) over two seconds after addition of 400 nM extension oligonucleotides. (Figure 13B) Reducing the connection between BtuB and the DNA origami modular device allowed the plug domain to refold and transition from an open channel back to a closed state. Breaks in the traces represent preparations for thoroughly mixing the cis compartment. Arrows on the graphs indicate where the extension oligonucleotides and the DTT reducing agent solutions are added.

[0071] Figure 14 represents the design of a DNA origami module. (Figure 14A) Schematic illustration of the DNA origami module comprising an extended element and a sleeve. (Figure 14B) The DNA origami module design with the caDNAno tool, the design consists of two domains, a long six-helix bundle extended element at the center topped with a head (left side on the caDNAno design), and a DNA origami sleeve folded concentrically around the extended element with an about 2nm gap between them, cross section right side on the figure 14B.

[0072] Figure 15 represents the DNA origami module (piston-cylinder) design and the folding results. (Figure 15A) The optimal folding conditions for the DNA origami module were evaluated by performing the folding with concentrations of 14 mM, 16 mM, 18 mM, 20 mM or 25 mM of magnesium (MgCls). M being the Molecular weight marker - 1000 bp. The resulting origami were separated on a 1% agarose gel with 1 1 mM of MgCh within the gel and the 0.5X TBE buffer. (Figure 15B) Exemplar TEM image of 97 nucleotides strand connector length DNA origami module folded in 18mM of MgCI2 after purification from agarose gel.

[0073] Figure 16 represents the DNA origami supports. (Figure 16A). The optimal folding condition for the DNA origami supports performed using concentrations of 14 mM, 16 mM, 18 mM, 19 mM or 20 mM of magnesium. M being the Molecular weight marker - 1000 bp. DNA origami supports were separated from excess staple oligonucleotides and evaluated in 1% agarose with 0.5XTBE and 1 1 mM of MgCI2. Uncropped and unprocessed scans of all the gels are provided in the source data. (Figure 16B) Exemplar TEM image of DNA origami support folded in 18mM of MgCI2 after purification from agarose gel.

[0074] Figure 17 represents the DNA origami modular device assembly. (Figure 17A) Complementary single strands on the sleeve and the heads of the DNA origami supports anneal together to (Figure 17B) adhere the supports to the sides facing the sleeve. (Figure 17C) DNA origami modular devices were tested using different molar ratios (1/4:1 - ¹ /z:1 - 1 :1 - 4:1 ) of DNA origami supports to DNA origami module and analyzed on 1% agarose gel in 0.5XTBE buffer and 11 mM of MgCI2. A ratio of 2:1 corresponding of DNA origami support: DNA origami module was sufficient to achieve the full ternary complex. M being the Molecular weight marker - 1000 bp. “Piston-Cylinder” being a control sample with the DNA origami module and “Landing Lag” being a control sample with the DNA origami supports. (Figure 17D) DNA origami modular device was tested for different incubation periods (2h; 4h; 10h; or 20 h) at 37°C then analyzed on 1% agarose gel in 0.5XTBE buffer and 1 1 mM of MgCI2. M being the Molecular weight marker - 1000 bp. “Piston-Cylinder” being a control sample with the DNA origami module and “Landing Lag” being a control sample with the DNA origami supports.

[0075] Figure 18 represents exemplar images of the assembly of the DNA origami modular device with side view (Figure 18A) and top view (Figure 18B).

[0076] Figure 19 represents the cholesterol functionalization of the DNA origami modular device. (Figure 19A) Cholesterol-functionalized oligonucleotides anneal on the underside of the bases of the DNA origami supports. (Figure 19B) Structure and sequence of the cholesterol-oligonucleotide. (Figure 19C) M (Molecular weight marker - 1000 bp); (lane 1 ) DNA origami supports with anchor elements without cholesterol-oligonucleotide or (lane 2) DNA origami supports with anchor elements incubated with 200nM of cholesterol- oligonucleotide for 30 minutes at 37°C. It is observed that the cholesterol-oligonucleotide blocks the migration of the DNA origami modular device. Samples were visualized on 1% agarose gel in 0.5XTBE buffer and 11 mM of MgCI2.

[0077] Figure 20 represents the resulting force, piconewton (pN), as a function of kpr spanning typical values for protein molecular systems. The DNA origami modular device is assembled with (1 ) a double strand of 97 nucleotides, (2) a double strand of 60 nucleotides, (3) a single strand of 97 nucleotides, or (3) a single strand of 60 nucleotides connector loops, respectively.

[0078] Figure 21 represents the synthesis of the RGD-oligonucleotide as set forth in the examples.

[0079] Figure 22 represents the functionalized end of the extended element on a distal position relating to the surface of interest (piston-cylinder tip). (Figure 22A) The six- helix bundle extremity of the extended element of the DNA origami module functionalized with three ligands. (Figure 22B) Cyclic RGD-conjugated oligonucleotides (left to the equal) to anneal on the end of the extended element on a distal position relating to the surface of interest.

[0080] Figure 23 represents the size exclusion of BtuBT3CHis-oligonucleotide. BtuBT3CHis-oligonucleotide was separated from excess oligonucleotide using a Superdex 200 HR 10/30 column. Fractions were analyzed by 4-20% SDS-PAGE. (Lane 1 ): BtuBT3CHis start material prior to conjugation with thiolated oligonucleotide.(Lanes 2 to 9): fractions 12 to 14 alternating with (+) and without (-) incubation with dithiothreitol (DTT) prior to migration on SDS-PAGE. [0081] Figure 24 represents histograms of the force applied by the DNA origami modular device. A simulation of the DNA origami module suspended above a membrane of a surface of interest with (Figure 24A) single-stranded, and (Figure 24B) double-stranded connector configurations (remotely activation). (Figure 24C) Average force extension profiles of the DNA origami module with single-stranded connectors and double-stranded Kpr(oxDNA) = 0.0057 (N/m)

[DESCRIPTION OF THE SEQUENCES]

[DETAILED DESCRIPTION]

Definitions [0082] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, may provide one of skill with a general dictionary of many of the terms used in this disclosure. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well- known and commonly used in the art. Methods are performed according to kits manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0083] Units, prefixes, and symbols are denoted in their Systeme International des Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0084] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents’ forms part of the common general knowledge in the art.

[0085] It is to be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid strand” includes a plurality of such nucleic acid strands, and so forth.

[0086] It is understood that aspects and embodiments of the present disclosure described herein include “having,” “comprising,” “consisting of,” and “consisting essentially of” aspects and embodiments. The words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of the stated element(s) (such as a composition of matter or a method step) but not the exclusion of any other elements. The term “consisting of” implies the inclusion of the stated element(s), to the exclusion of any additional elements. The term “consisting essentially of” implies the inclusion of the stated elements, and possibly other element(s) where the other element(s) do not materially affect the basic and novel characteristic(s) of the disclosure. It is understood that the different embodiments of the disclosure using the term “comprising” or equivalent cover the embodiments where this term is replaced with “consisting of” or “consisting essentially of”.

[0087] The term, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0088] The term “approximately” or "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some embodiments, the term indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%.

[0089] Within the disclosure, the term ‘significantly” used with respect to change intends to mean that the observed change is noticeable and/or it has a statistic meaning.

[0090] Within the disclosure, the term “substantially” used in conjunction with a feature of the disclosure intends to define a set of embodiments related to this feature which are largely but not wholly similar to this feature.

[0091] The term "nucleic acid strand" as used herein intends to refer to a polymer composed of nucleotides, such as deoxyribonucleotides or ribonucleotides. The term “nucleic acid” may generally encompass "ribonucleic acid" and "deoxyribonucleic acid". The expressions and terms “nucleic acid”, “nucleic acid sequence”, “nucleic acid strand”, or “oligonucleotide” are used interchangeably.

[0092] The term “nucleotide” intends to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Generally, a nucleotide may be an adenine (A), a Thymine (T), an uracil (U), a cytosine (C), or a guanine (G).

[0093] "Ribonucleic acid" and "RNA" as used herein intends to refer to a polymer composed of ribonucleotides. A ribonucleotide is a nucleotide containing ribose as its pentose component. The nucleotide bases for ribonucleotides are adenine (A), guanine (G), cytosine (C), or uracil (U).

[0094] "Deoxyribonucleic acid" and "DNA" intends to refer to a polymer composed of deoxyribonucleotides. A deoxyribonucleotide is a nucleotide that contains deoxyribose. The nucleotide bases for ribonucleotides are adenine (A), guanine (G), cytosine (C), or thymine (T).

[0095] As used herein, “nucleic acid origami” intends refer to the result of the folding of one or more long “scaffold” nucleic acid strands into a particular shape using a plurality of rationally designed “staple” nucleic acid strands. In particular, the scaffold nucleic acid strand may be folded into one or more helices of nucleic acids using a plurality of staple strands, thereby forming a particular shape. A nucleic acid origami encompasses a DNA origami, a RNA origami or a combination thereof.

[0096] As used herein, “helix” refers to the paired regions of a scaffold nucleic acid strand with one or more staple nucleic acid strand(s) forming the helix. In particular, the helix is a double-helix of nucleic acids.

[0097] As used herein the expressions “nucleic acid origami modular device”, “modular device” and simply “device” may be used interchangeably and intend refer to the result of the attachment of different components, or module, each being a separate nucleic acid origami.

[0098] As used herein, the term “modular” means that a module is separable from the remainder of the device and can be replaced by another module having substantially similar shape, dimension, activity and/or other surface features including, but not limited to, the position and elements allowing the attachment or the binding. By way of example, a nucleic acid origami support attaches to, but is not part of, the nucleic acid origami module. Thus, the nucleic acid origami module and the nucleic acid origami support are independently produced and assembled to obtain a nucleic acid origami modular device of the disclosure.

[0099] The terms “attaching” or “attach” used in connection with the first component and second component of the nucleic acid origami modular device of the disclosure or in connection with the nucleic acid origami modular device and the surface of interest intends to refer the fastening, usually in a non-covalent manner, of the first and second components together or of the nucleic acid origami modular device to the surface of interest. For instance, the first and second components may be attached by hybridization of staple strands, and the nucleic acid origami modular device may be attached to the surface of a cell membrane by means of moieties, such as ligands, for instance protein ligands or cholesterol groups, from anchor members inserted the lipid bilayers of the cell membrane.

[0100] The term "scaffold strand" as used herein intends to refer to a long nucleic acid, e.g., DNA, strand, for example about 7560 bases long, that may be folded into a particular shape using a plurality of rationally designed "staple strands". There is no principal limit to the length of scaffold strand; it all depends on the size of device to build, so the scaffold strand could have any length between 15 and 20 000 nucleotides. The nucleic acid sequences of the staple strands are designed such that they hybridize to particular portions of the scaffold strands and, in doing so, force the scaffold strands into a particular shape.

[0101] Methods useful in the making of DNA origami structures can be found, e.g., in Rothemund, P.W., Nature 440:297-302 (2006); Douglas et al., Nature 459:414-418 (2009); Dietz et aL, Science 325:725-730 (2009); and US Patent Publication Nos. 2007/0117109, 2008/0287668, 20100069621 and 2010/0216978. Staple design can be facilitated using, e.g., CADnano software, available at http://www.cadnano.org

[0102] “Hybridization” or “hybridizing” intend to refer to the base pairing or the formation of a duplex between nucleotides, such as, for instance, between two strands of complementary or substantially complementary nucleic acid. Complementary nucleotides are, generally, adenine (A) and Thymine (T)/uracil (U), or cytosine (C) and guanine (G). Two single-stranded RNA or DNA molecules are said to be complementary or substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Alternatively, a substantial complementarity exists when a nucleic acid strand hybridizes under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementarity over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementarity. Selective hybridization conditions are known in the art. [0103] “Surface of interest” intends to refer to the exterior or upper boundary of an object or body. A surface may be biological or non-biological, such as an artificial surface. A biological surface may be a surface of a biological tissue or of a cell, such as a prokaryote or an eukaryote cell. The surface of a cell is the exterior boundary of the cell membrane.

[0104] The terms “interacting” or “interact”, or the like, used in connection with a nucleic acid module and a surface of interest intend to refer to an action of the nucleic acid module upon the surface which results in a force being exerted on the surface, and possibly the induction of a mechanical constraint.

[0105] The term “force” when used in connection with a result of an interaction, in particular a physical interaction, of the nucleic acid origami module with a surface of interest intends to refer to the magnitude and direction of the change imparted by the nucleic acid module to the surface of interest. The change may be a mechanical constraint, such as a pression or a traction, induced by the nucleic acid module on the surface. In particular, the force may be a mechanical force. A pression or a traction may be induced by the nucleic acid module with a physical interaction on the surface.

[0106] As used herein the expression "mechanical constraint" used with regard to the surface of interest intends to refer a result of force, in particular a mechanical force, exerted on that surface. The result may be a movement or a deformation of at least a part of the surface or of an element of said surface. For example, a mechanical constraint may result in a displacement or an extension of a protein present on or in the surface of interest. A mechanical constraint in the context of the present disclosure may be induced the nucleic acid module with a physical interaction on the surface.

[0107] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. [0109] The list of sources, ingredients, and components as described hereinafter are listed such that combinations and mixtures thereof are also contemplated and within the scope herein.

[0110] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0111] All lists of items, such as, for example, lists of ingredients, are intended to and should be interpreted as Markush groups. Thus, all lists can be read and interpreted as items “selected from the group consisting of’ the list of items “and combinations and mixtures thereof.”

[0112] Referenced herein may be trade names for components including various ingredients utilized in the present disclosure. The inventors herein do not intend to be limited by materials under any particular trade name. Equivalent materials (e.g., those obtained from a different source under a different name or reference number) to those referenced by trade name may be substituted and utilized in the descriptions herein.

Nucleic acid origami modular device

[0113] Nucleic acid origami structures incorporate nucleic acids as a building material to make nanoscale shapes. In general, the nucleic acid origami process involves the folding of one or more long single-stranded nucleic acid, named “scaffold strand”, into a particular shape using a plurality of specific complementary designed single-stranded nucleic acid, named “staple strand”.

[0114] The nucleic acid sequences of the staple strands are designed such that they hybridize to particular portions of the scaffold strands and allow the scaffold strands folding into a particular shape. Methods useful in the making of acid nucleic origami structures can be found, for example, in Douglas et aL, Nature 459:414-418 (2009); and U.S. Pat. App. Pub. Nos. 2013/0224859, 2008/0287668, 2010/0069621 and 2010/0216978. [0115] Further, the shape of the device may be defined according to the purpose of the device and may be obtained by designing the set of staple strands allowing shaping a scaffold strand into the intended device. Design of staple strands can be carried with various computer programs known in the art, such as CADnano software, available at http://www.cadnano.org.

[0116] The nucleic acid scaffold strands and the nucleic acid staple strands of the nucleic acid origami modular device are selected such that the nucleic acid origami modular device has a first module and a second module, wherein the first module is at least one nucleic acid origami support and the second module is a nucleic acid origami module, configured to be an actuator (nucleic acid origami actuator), attached to the at least one nucleic acid origami support, as described below.

[0117] A nucleic acid origami modular device of the disclosure comprising a first component and a second component. The first and second components are the modules of the modular device. Each of the first and second component comprises a nucleic acid scaffold strand and a plurality of nucleic acid staple strands. The first component and second component are attached together by hybridization of at least two staple strands. The first component is at least one nucleic acid origami support, and the second component is a nucleic acid origami module (an actuator). The nucleic acid origami support comprises at least one anchor element for attaching the nucleic acid origami modular device to a surface of interest. The nucleic acid origami module is configured for interacting with said surface of interest.

[0118] The first component may be comprised of a plurality of nucleic acid origami supports. The first component may be comprised of at least two, three, four, five or more nucleic acid origami supports. The first component may be comprised of two nucleic acid origami supports. The first component may consist of two nucleic acid origami supports.

[0119] In some embodiments, a nucleic acid origami modular device may comprise from one to twenty nucleic acid origami supports attached to the nucleic acid origami module.

[0120] A nucleic acid origami modular device may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen and twenty nucleic acid origami supports attached to the nucleic acid origami module. [0121] A nucleic acid origami modular device of the disclosure may comprise from at least one to at least six nucleic acid origami supports attached to the nucleic acid origami module.

[0122] A nucleic acid origami modular device of the disclosure may comprise from at least one to at least four nucleic acid origami supports attached to the nucleic acid origami module.

[0123] A nucleic acid origami modular device of the disclosure may comprise two nucleic acid origami supports attached to the nucleic acid origami module.

[0124] The first and the second components are arranged to cooperate together for exerting a force, in particular a mechanical force, on the surface of interest.

[0125] The first component which is a nucleic acid origami support with anchor members allows attaching the nucleic acid origami modular device. The second component, the nucleic acid origami module, is an actuator. The nucleic acid origami module comprises binding moieties able to interact with complementary moieties present at the surface of interest. The nucleic acid origami module is configured to move relative to the nucleic acid origami support and to interact with the surface of interest. The nucleic acid origami support being attached to the surface of interest, it may oppose to the nucleic acid origami module movement so that the movement of the nucleic acid origami module is translated into a force exerted at the surface of interest, in particular at the point of interaction between the binding moieties and the complementary moieties. The exerted force may induce a mechanical constraint at the surface of interest, such as a pression, a traction, a twist, or a stretch. Together, the sequence of steps described herein, attachment of the nucleic acid origami support to the surface of interest, movement of the nucleic acid origami module relative to the nucleic acid origami support, interaction of the nucleic acid origami module with the surface, opposition (or resistance) of the nucleic acid origami support to the nucleic acid origami module, defines the cooperation between the first and second component for exerting a force on the surface of interest.

[0126] The nucleic acid origami modular device allows exerting and measuring forces at a surface of interest in the range of picoNewton.

[0127] A nucleic acid origami modular device of the disclosure may be comprised of at least two single scaffold strands and a plurality of staple strands. For example, one scaffold strand forms a nucleic acid origami module, and the other scaffold strand forms a nucleic acid origami support. [0128] Since a first component of the nucleic acid modular device may be comprised of a plurality of nucleic acid origami supports, there may be as much as nucleic acid scaffold strands than nucleic acid origami supports.

[0129] For example, for a first component being comprised of at least two nucleic acid origami supports, there are two nucleic acid scaffold strands, each being folded with a set of nucleic acid staple strands.

[0130] In some embodiments, a nucleic acid origami modular device of the disclosure may comprise three single scaffold strands: one for the nucleic acid origami module and two for two nucleic acid origami supports, each scaffold strand being folded in the corresponding origami with the suitable set of staple strands.

[0131] The nucleic acid scaffold strands of a nucleic acid origami modular device may be identical or different. In some embodiments, the nucleic acid scaffold strands are identical.

[0132] A sequence of a nucleic acid scaffold strand suitable for a nucleic acid origami modular device of the disclosure may have at least about 70% to about 100% identity with SEQ ID NO: 408.

[0133] A sequence of a nucleic acid scaffold strand may have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identity with SEQ ID NO: 408.

[0134] In some embodiments, a sequence of a nucleic acid scaffold strand may have a nucleic acid sequence as set forth in SEQ ID NO: 408.

[0135] The sequences of the nucleic acid staple strands suitable for a nucleic acid origami modular device of the disclosure may have at least about 70% to about 100% identity with SEQ ID NOs: 1 to 387.

[0136] The sequences of the nucleic acid staple strands may have at least about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identity with SEQ ID NOs: 1 to 387.

[0137] In some embodiments, the nucleic acid sequences of the nucleic acid staple strands may have a nucleic acid sequence as set forth in SEQ ID NOs: 1 to 387.

[0138] A nucleic acid suitable for a nucleic acid origami modular device of the disclosure may a DNA, an RNA, or a nucleic acid comprising modified nucleotide, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleic acids may be interrupted by non-nucleotide components. A nucleic acid may be further modified after polymerization, such as by conjugation with a labeling component. The term “complement of a nucleic acid” denotes a nucleic acid molecule having a complementary base sequence and reverse orientation as compared to a reference sequence, such that it could hybridize with a reference sequence with complete fidelity. “Recombinant” as applied to a nucleic acid means that the nucleic acid is the product of various combinations of in vitro cloning, restriction and/or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell. A nucleic acid may comprise conventional phosphodiester bonds and/or non-conventional bonds, e.g., an amide bond, such as found in peptide nucleic acids (PNA).

[0139] In some embodiments, the nucleic acid of the first and/or second component may be a DNA or an RNA.

[0140] In some embodiments, the nucleic acid of the first and second components may be a DNA.

[0141] The nucleic acid used as a scaffold strand for the first and second components may be of any suitable length.

[0142] The nucleic acid used as a scaffold strand for the first component may be the same or different from the nucleic acid used as scaffold strand for the second component. In some embodiments, the nucleic acids used as scaffold strands for the first and the second component are the same.

[0143] The nucleic acids used for the staple strands may be of any length. The nucleic acids used for the staple strands hybridize to scaffold strands to fold the scaffold strands into, respectively, nucleic acid origami module and nucleic acid origami support(s).

[0144] The sets of nucleic acids used staple strands are selected according to the origami folding to achieve.

Nucleic acid origami support

[0145] A nucleic acid origami modular device of the disclosure comprising a first component and a second component. The first component is a module of the nucleic acid origami modular device of the disclosure. A first component may be at least one nucleic acid origami support. In some embodiments, a first component may be at least two nucleic acid origami supports.

[0146] When a first component comprises more than one nucleic acid origami support, all the origami supports constitutes together the module defined by the first component. They each can be considered as sub-module of the main module defined by the first component.

[0147] When a first component comprises more than one nucleic acid origami support, all the origami supports may be identical or different. In some embodiments, when a first component comprises more than one nucleic acid origami support, all the origami supports may be identical.

[0148] A nucleic acid origami support comprises a scaffold strand and a plurality of staple strands. At least one staple strand may be suitable for hybridizing to a sequence of a nucleic acid of the second component. At least one staple strand may be suitable for being an anchor element suitable for attaching the nucleic acid origami support, and therefore, the nucleic acid origami modular device, to a surface of interest.

[0149] In some embodiments, a sequence of the nucleic acid scaffold strand may be suitable for hybridizing to a sequence of a nucleic acid of the second component.

[0150] In some embodiments, a sequence of a nucleic acid staple strand may be suitable for hybridizing to a sequence of a nucleic acid staple strand of the second component.

[0151] A nucleic acid origami support may comprise a head for attaching the nucleic acid origami support to the nucleic acid origami module, and at least one extended member projecting from said head, said extended member being connected by one end to said head and by the other end to at least one base, said base comprising said at least one anchor element.

[0152] A nucleic acid origami support may comprise at least one, two, three or more extended member(s).

[0153] In some embodiments, a nucleic acid origami support may comprise at least one, two, or three extended member(s).

[0154] In some embodiments, a nucleic acid origami support may comprise two extended members. [0155] A nucleic acid origami support may comprise a head for attaching the nucleic acid origami support to the nucleic acid origami module, and at least two extended members projecting from said head, said each extended members being connected by one end to said head and by the other end to at least one base, said base comprising said at least one anchor element.

[0156] In some embodiments, an extended member may be connected to one, two, three, or more base(s).

[0157] In some embodiments, an extended member may be connected to one, two, or three base(s).

[0158] In some embodiments, an extended member may be connected to one base.

[0159] A base may comprise from one to twelve anchor elements.

[0160] In some embodiments, a base may comprise from one to eight anchor elements. In some embodiments, a base may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve anchor elements.

[0161] In some embodiments, a base may comprise eight anchor elements.

[0162] The anchor element(s) are arranged on the base for being oriented towards the surface of interest. Therefore, the anchor element can attach to the surface of interest, and, consequently, attach the nucleic acid modular device to the surface of interest.

[0163] The anchor element may be a nucleic acid staple strand. The nucleic acid staple strand of the anchor element is complementary to a sequence of a nucleic acid of the base. It may be complementary to a nucleic acid staple strand of the base, or it may be complementary to a sequence of the nucleic acid scaffold of the nucleic acid origami support folded into a base. In some embodiments, a nucleic acid staple strand of the anchor element is complementary to a sequence of the nucleic acid scaffold of the nucleic acid origami support folded into a base.

[0164] In some embodiments, a nucleic acid sequence of an anchor element of a nucleic acid origami support may comprise or consist in a nucleic acid sequence having at least about 70% to about 100% identity with a nucleic acid sequence selected from SEQ ID NOs: 372 to 387.

[0165] A nucleic acid sequence of an anchor element may have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identity with a nucleic acid sequence selected from SEQ ID NOs: 372 to 387.

[0166] A nucleic acid sequence of an anchor element may have a nucleic acid sequence as set forth in any one of SEQ ID NOs: 372 to 387.

[0167] In some preferred embodiments, a nucleic acid origami support may comprise sixteen anchor elements arranged on two bases. In these embodiments, the nucleic acid sequences of the sixteen anchor elements may have nucleic acid sequences as set forth in SEQ ID NOs: 372 to 387.

[0168] The nucleic acid staple strand of the anchor element may be functionalized (or conjugated) with at least one moiety suitable to adhere to or insert in said surface of interest. The anchor element(s) are arranged on the base for having the moiety(ies) being oriented towards the surface of interest.

[0169] The moiety may be suitable to adhere to a target protein on the surface of interest.

[0170] An anchor element may comprise at least one ligand-conjugated oligonucleotide.

[0171] A ligand may be a lipid, an antibody, a nanobody, or an antigen-binding fragments thereof.

[0172] In some embodiments, a ligand may be an antibody or a nanobody.

[0173] The term "antibody" used herein intends to refer to an isolated or recombinant immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen, such as an antigen present on the membrane of a cell of interest. As such, the term antibody encompasses not only whole antibody molecules, but also antigen-binding fragments thereof such as “nanobody” as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulphide bonds and each heavy chain is linked to a light chain by a disulphide bond. There are two types of light chain, lambda (1 ) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain

(VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1 -L, CDR2-L, L- CDR3-L and CDR1 -H CDR2-H, CDR3-H, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.

[0174] As used herein, the term "nanobody" or “single-domain antibody” means an antibody fragment derived from a class of camelid antibodies having a small size (1/10 the size of conventional antibodies). According to some embodiments, the nanobodies are suitable for genetic engineering, and further they are able to bind their antigens with a single variable heavy chain.

[0175] An antibody suitable as ligand may be an antibody able to bind to a target protein on a surface of interest.

[0176] A nanobody suitable as ligand may be capable to bind to a target protein on a surface of interest, in particular a nanobody capable to bind to a receptor on a surface of interest.

[0177] In some embodiments, a nanobody suitable as ligand may be capable to bind to an integrin receptor, for instance the nanobody hCD1 1 bNb1 (Rasmus et aL, J Biol Chem, 2022, article in press, 102168 - https://doi.org/10.1016/jjbc.2022.102168).

[0178] In some embodiments, a ligand may be a lipid.

[0179] In some embodiments, a lipid may be chosen among a fatty acid, a glycerolipid, a glycerophospholipid, a sphingolipid, a phosphatidylcholine, a phosphatidylethanolamine, a sphingomyelin (SM), or a ceramide, a sterol, a prenol, a saccharolipid, a polyketide, or a combination thereof.

[0180] A phosphatidylcholine may be DSPC (1 ,2-distearoyl-sn-glycero-3- phosphocholine), DPPC (1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1 ,2- dimyristoyl-sn-glycero-3-phosphocholine), POPC (1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine), DOPC (1 ,2-dioleoyl-sn-glycero-3-phosphocholine).

[0181] A phosphatidylethanolamine may be DOPE (1 ,2-dioleyl-sn-glycero-3- phosphoethanolamine), DPPE (1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DSPE (1 ,2-distearoyl-s/i- glycero-3-phosphoethanolamine), DLPE (1 ,2-dilauroyl-SM-glycero-3- phosphoethanolamine), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, or I- stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE).

[0182] A sterol may be chosen among cholesterol, ergosterol, a hopanoid, a hydroxysteroid, a phytosterol, a steroid, zoosterolor, ergosterol, desmosterol (3B-hydroxy- 5,24-cholestadiene), stigmasterol (stigmasta-5,22-dien-3-ol), lanosterol (8,24-lanostadien- 3b-ol), 7-dehydrocholesterol (A5,7-cholesterol), dihydrolanosterol (24,25- dihydrolanosterol), zymosterol (5a-cholesta-8,24-dien-3B-ol), lathosterol (5a-cholest-7-en- 3B-ol), diosgenin ((3p,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3B-ol), campestanol (5a-campestan-3b-ol), 24- methylene cholesterol (5,24(28)-cholestadien-24-methylen-3B-ol); BHEM-Cholesterol (2- (((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-meth ylheptan-2-yl)- 2,3,4,7,8,9,10,11 ,12,13,14,15,16,17-tetradecahydro- 1 H-cyclopenta[a]50henanthrene-3- yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethan -1 -aminium bromide); and combinations thereof.

[0183] In some preferred embodiments, an anchor element may comprise at least one cholesterol-conjugated oligonucleotide.

[0184] A nucleic acid sequence of a ligand-conjugated oligonucleotide may comprise or consist in a nucleic acid sequence having at least about 70% to about 100% identity with a nucleic acid sequence selected from SEQ ID NOs: 356 to 371 .

[0185] A nucleic acid sequence of a ligand-conjugated oligonucleotide may have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identity with a nucleic acid sequence selected from SEQ ID NOs: 356 to 371. [0186] A nucleic acid sequence of a ligand-conjugated oligonucleotide may have a nucleic acid sequence as set forth in any one of SEQ ID NOs: 356 to 371 .

[0187] In some preferred embodiments, a nucleic acid origami support may comprise sixteen ligand-conjugated oligonucleotides arranged on two bases. In these embodiments, the nucleic acid sequences of the sixteen ligand-conjugated oligonucleotides may have nucleic acid sequences as set forth in SEQ ID NOs: 356 to 371 .

[0188] As indicated, a nucleic acid origami support comprises at least one nucleic acid scaffold strand and a plurality of nucleic acid staple strands. The staple strands are designed to hybridize to the scaffold strand to fold it into a nucleic acid origami support.

[0189] The strands are single-strand nucleic acids.

[0190] In some embodiments, a nucleic acid origami support may comprise one nucleic acid scaffold strand.

[0191] In some embodiments, a nucleic acid origami support may comprise a plurality of nucleic acid scaffold strands folded into the different parts of the support: head, extended member(s) and base(s), the different parts being held together in a nucleic acid origami support by hybridization.

[0192] In some embodiments, a nucleic acid origami support may comprise three nucleic acid scaffold strands each forming independently a head, and two extended members prolonged by a base.

[0193] In some embodiments, a nucleic acid origami support may comprise a single nucleic acid scaffold strand folded into the support with a plurality of nucleic acid staple strands.

[0194] A scaffold strand may be of a length of about 15 to about 20 000 nucleotides (nt).

[0195] A scaffold strand may be of a length of about 50 to about 19 000 nucleotides (nt), or about 100 to about 18 500 nt, or about 1 000 to about 18 000 nt, or about 1 500 to about 17 000 nt, or about 2 000 to about 16 000 nt, or about 3 000 to about 15 000 nt, or about 4 000 to about 14 000 nt, or about 4 500 to about 13 000 nt, or about 5 000 to about 12 000 nt, about 5 500 to about 1 1 000 nt, or about 6 000 to about 10 000 nt, or about 6 500 to about 9 000 nt, about 6 800 to about 8 500 nt, or about 7 000 to about 8 400 nt, or about 7 200 to about 8 200 nt, or about 7 400 to about 8 100 nt, or about 7 450 to about 8 000 nt, or about 7 500 to about 7 800 nt, or about 7 500 to about 7 600 nt. [0196] A single scaffold strand may be of a length of about 7 560 nt.

[0197] In some embodiments, the sequence of the nucleic acid scaffold strand of a nucleic acid origami support may comprise or consist in a sequence having at least about 70% to about 100% identity with SEQ ID NO: 408.

[0198] The term “identity” in the context of two or more nucleic acids sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection (for example NCBI web site or the like). This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods. Identity (e.g., percent homology) may be determined using various known sequence comparison tools, such as any homology comparison software computing a pairwise sequence alignment, including for example, the Blast software of the National Center of Biotechnology Information (NCBI), by using default parameters. The identity is a global identity, i.e., an identity over the entire nucleic acid sequences and not over portions thereof. Pairwise global alignment was defined by Needleman et al., Journal of Molecular Biology, 1970, pages 443-53, volume 48).

[0199] A sequence of the nucleic acid scaffold strand of an origami support may comprise or consist in a sequence having at least about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with SEQ ID NO: 408.

[0200] In some embodiments, a sequence of the nucleic acid scaffold strand of an origami support may consist in the nucleic acid sequence SEQ ID NO: 408.

[0201] A staple strand for an origami support may be a nucleic acid of a length of about 12 to about 100 nt.

[0202] A staple strand may be a nucleic acid of a length of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64,

65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88,

89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or about 100 nt.

[0203] A staple strand may be a nucleic acid of a length of about 10 to about 100 nt.

[0204] A staple strand may be a nucleic acid of a length of about 20 to about 70 nt.

[0205] The number of staple strands to form a nucleic acid origami support may be determined by considering the number of nucleotides in a nucleic acid scaffold strand and the number of nucleotides in the smallest nucleic acid staple strand. Specifically, the maximum number of staple strands to be used in a nucleic acid origami of the disclosure is the ratio of the number of nucleotides in a nucleic acid scaffold strand to the number of nucleotides in the smallest nucleic acid staple strand.

[0206] The number of staple strands to form a nucleic acid origami support may range from 10 to about 1000 staple strands.

[0207] The number of staple strands to form a nucleic acid origami support may range from about 20 to about 700 staple strands, or from about 50 to about 600 staples strands, or from about 100 to about 450, or from about 150 to about 400 staples strands.

[0208] The sequences of the nucleic acid staple strands of an origami support may comprise or consist in a sequence having at least about 70% to about 100% sequence identity with a sequence selected from SEQ ID NOs: 159 to 347.

[0209] The sequences of the nucleic acid staple strands of an origami support may comprise or consist in a sequence having at least about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with a sequence selected from SEQ ID NOs: 159 to 347.

[0210] The sequences of the nucleic acid staple strands of an origami support may consist in the sequences SEQ ID NOs: 159 to 347.

[0211] As indicated, a nucleic acid origami support may comprise a head for attaching the nucleic acid origami support to the nucleic acid origami module, and at least two extended members projecting from said head, said each extended members being connected by one end to said head and by the other end to at least one base, said base comprising said at least one anchor element. [0212] A base of a nucleic acid origami support may be comprised of a nucleic acid scaffold strand folded in at least two helices.

[0213] A base may be comprised of a nucleic acid scaffold strand folded in two to fifteen helices.

[0214] A base may be comprised of a nucleic acid scaffold strand folded in 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 helices.

[0215] In some embodiments, a base of a nucleic acid origami support may be comprised of a nucleic acid scaffold strand folded in twelve helices.

[0216] A base of a nucleic acid origami support may be comprised of a nucleic acid scaffold strand folded in at least two bundles of six-helix bundle (6-HB).

[0217] A base may be comprised of a nucleic acid scaffold strand folded in two to five bundles of six-helix bundle (6-HB).

[0218] A base may be comprised of a nucleic acid scaffold strand folded in two, three, four or five bundles of six-helix bundle (6-HB).

[0219] In some embodiments, a base may be comprised of a nucleic acid scaffold strand folded in two bundles of six-helix bundle (6-HB).

[0220] An extended member of a nucleic acid origami support may be comprised of a nucleic acid scaffold strand folded in at least six helices.

[0221] An extended member may be comprised of a nucleic acid scaffold strand folded in six to thirty-six helices.

[0222] An extended member may be comprised of a nucleic acid scaffold strand folded in 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, 31 , 32, 33, 34, 35 or 36 helices.

[0223] In some embodiments, an extended member of a nucleic acid origami support may be comprised of a nucleic acid scaffold strand folded in twelve helices.

[0224] An extended member of a nucleic acid origami support may be comprised of a nucleic acid scaffold strand folded in at least one bundle of a six-helix bundle (6-HB).

[0225] An extended member may be comprised of a nucleic acid scaffold strand folded in one to ten bundles of six-helix bundle (6-HB).

[0226] An extended member may be comprised of a nucleic acid scaffold strand folded in one, two, three, four, five, six, seven, eight, night or ten bundles of six-helix bundle (6-HB). [0227] In some embodiments, an extended member may be comprised of a nucleic acid scaffold strand folded in two bundles of six-helix bundle (6-HB).

[0228] A head of a nucleic acid origami support may be comprised of a nucleic acid scaffold strand folded in at least twelve helices.

[0229] A head may be comprised of a nucleic acid scaffold strand folded in twelve to fifty helices.

[0230] A head may be comprised of a nucleic acid scaffold strand folded in 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 helices.

[0231] A head of a nucleic acid origami support may be comprised of a nucleic acid scaffold strand folded in at least two bundles of six-helix bundle (6-HB).

[0232] A head may be comprised of a nucleic acid scaffold strand folded in two to fifteen bundles of six-helix bundle (6-HB).

[0233] A head may be comprised of a nucleic acid scaffold strand folded in two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen bundles of six-helix bundle (6-HB) of nucleic acid.

[0234] A head of a nucleic acid origami support may be of a length ranging from about 10 nm to about 60 nm.

[0235] A head may be of a length of about 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm or about 60 nm.

[0236] In some embodiments, a head of a nucleic acid origami support may be of a length of 30 nm.

[0237] In some embodiments, the head of a nucleic acid origami support may comprise at least one sequence of nucleic acid complementary to a sequence of the nucleic acid origami module.

[0238] The head may comprise from one to eight sequences of nucleic acids complementary to one to eight sequences of the nucleic acid origami module. [0239] In some embodiments, the head of the nucleic acid origami support may comprise eight sequences of nucleic acids complementary to eight sequences of the nucleic acid origami module.

[0240] The head of the nucleic acid origami support may be arranged to have the sequences of nucleic acids complementary to sequences of the nucleic acid origami module at close proximity from one to each other.

[0241] A sequence of a nucleic acid complementary to a sequence of the nucleic acid origami module may comprise or consist in a sequence having at least about 70% to about 100% identity with a sequence selected from SEQ ID NOs: 348 to 355.

[0242] A sequence of a nucleic acid complementary to a sequence of the nucleic acid origami module may comprise or consist in a sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% identity with a sequence selected from SEQ ID NOs: 348 to 355.

[0243] A sequence of a nucleic acid complementary to a sequence of the nucleic acid origami module may consist in a sequence selected from SEQ ID NOs: 348 to 355.

[0244] A base may be connected to or may a prolongation of an extended member. A base connecting or prolonging an extended member will be referred as the base of said extended member (an extended member and its base).

[0245] A base may project along a longitudinal axe perpendicular or substantially perpendicular to the longitudinal axe of the extended member.

[0246] In embodiments wherein a support comprises at least two extended members, and therefore at least two bases, each longitudinal axe of each base is coplanar relative to each other. The longitudinal axes of the two bases may be arranged together to form an angle ranging from about 30° to about 110°.

[0247] The longitudinal axes of two bases may be coplanar and may be arranged together to form an angle of about 30°, 31 °, 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, 40°, 41 °, 42°, 43°, 44°, 45°, 46°, 47°, 48°, 49°, 50°, 51 °, 52°, 53°, 54°, 55°, 56°, 57°, 58°, 59°, 60°,

61 °, 62°, 63°, 64°, 65°, 66°, 67°, 68°, 69°, 70°, 71 °, 72°, 73°, 74°, 75°, 76°, 77°, 78°, 79°,

80°, 81 °, 82°, 83°, 84°, 85°, 86°, 87°, 88°, 89°, 90°, 91 °, 92°, 93°, 94°, 95°, 96°, 97°, 98°,

99°, 100°, 101 °, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, or of about 110°.

[0248] In some embodiments, the longitudinal axes of two bases may be coplanar and may be arranged together to form an angle of about 45°. [0249] In some embodiments, a base may be of a length ranging from about 1 nanometer (nm) to about 100 nm.

[0250] A base may be of a length of about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm,

32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm,

44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm,

56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm,

68 nm, 69 nm, 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm,

80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm, 90 nm, 91 nm,

92 nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 or of about 100 nm.

[0251] In some embodiments, a base may be of a length of about 30 nm.

[0252] A head of a nucleic acid origami support may comprise at least two extended members projecting therefrom.

[0253] The two extended members comprise each a longitudinal axe extending perpendicular to the plan of a surface of interest to which a nucleic acid origami modular device of the disclosure is intended to be attached to.

[0254] The longitudinal axes may be parallel or substantially parallel one to each other.

[0255] An extended member and its base may be arranged one relative to the other to form an angle ranging from about 70° to about 1 10°.

[0256] The longitudinal axe of an extended member may be coplanar with the longitudinal axe of its base, and the axes may form together an angle ranging from about 70° to about 1 10°.

[0257] The angle may be of about 70°, 71 °, 72°, 73°, 74°, 75°, 76°, 77°, 78°, 79°, 80°, 81 °, 82°, 83°, 84°, 85°, 86°, 87°, 88°, 89°, 90°, 91 °, 92°, 93°, 94°, 95°, 96°, 97°, 98°, 99°, 100°, 101 °, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, or of about 110°.

[0258] In some embodiments, the angle may be of about 90°.

[0259] In some embodiments, an extended member and its base may form together a shape of a L.

[0260] In some embodiments, an extended member may be of a length ranging from about 20 nm to about 150 nm or more. [0261] An extended member may be of a length of about 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, 50 nm or more, 55 nm or more, 60 nm or more, 65 nm or more, 70 nm or more, 75 nm or more, 80 nm or more, 85 nm or more, 90 nm or more, 95 nm or more, 100 nm or more, 105 nm or more, 110 nm or more, 115 nm or more, 120 nm or more, 125 nm or more, 130 nm or more, 135 nm or more, 140 nm or more, 145 nm or more, or of about 150 nm or more.

[0262] In some embodiments, an extended member may be of a length of about 55 nm.

[0263] In some embodiments, an extended member and its base may be further connected together by at least one nucleic acid connector strand.

[0264] The number of nucleic acid connector strands may depend on the number of helices comprised in a base of a nucleic acid origami support, in particular the number of helices facing the extended member (a base is comprised of at least two sides or faces, one side bearing the anchor element(s) and facing the surface of interest, and one side, opposite to the side facing the surface of interest, and facing the extended member). The base may be connected to the extended member by means of its helix/ces facing the extended member. Also, the extended member may be connected to the base by means of its helix/ces facing the base.

[0265] The number of nucleic acid connector strands may be adapted by the skilled person in the art to strengthen the connection between the base and the extended member, considering the number of helices of the base and/or the number of helices of the extended member which can be effectively used. In some embodiments, the number of nucleic acid connector strand may be sufficient to define an angle of about 90° between the longitudinal axe of an extended member with the longitudinal axe of its base.

[0266] In some embodiments, an extended member and its base may be further connected together by one to thirty nucleic acid connector strand(s).

[0267] In some embodiments, an extended member and its base may be further connected together by one to twenty nucleic acid connector strand(s), or one to ten nucleic acid connector strand(s), or one to five nucleic acid connector strand(s).

[0268] In some embodiments, an extended member and its base may be further connected together by one nucleic acid connector strand. [0269] In some embodiments, an extended member and its base may be further connected together by two nucleic acid connector strands.

[0270] In some embodiments, the nucleic acid connector strand may be a singlestranded nucleic acid.

[0271] In some embodiments, the nucleic acid connector strand may be a doublestranded nucleic acid.

[0272] In some embodiments, the nucleic acid connector strand may be a doublestranded DNA.

[0273] In some embodiments, the nucleic acid connector strand may connect a side of a base, opposite to the side bearing the anchor element(s), to a side of its extended member facing this side of the base.

[0274] In some embodiments, an extended member and its base may not be further connected together by a nucleic acid connector strand.

Nucleic acid origami module

[0275] A nucleic acid origami modular device of the disclosure comprising a first component and a second component. The second component is a module of the nucleic acid origami modular device of the disclosure.

[0276] The nucleic acid origami module may be an actuator.

[0277] The nucleic acid origami module is configured to move relative to the nucleic acid origami support and to interact with the surface of interest.

[0278] The nucleic acid origami module and the nucleic acid origami support(s) are arranged to cooperate together for exerting a force on a surface of interest. The exerted force may induce a mechanical constraint at the surface of interest, such as a pression, a traction, a twist, or a stretch.

[0279] A nucleic acid origami module comprises a nucleic acid scaffold strand and a plurality of nucleic acid staple strands. At least one staple strand may be suitable for hybridizing to a sequence of a nucleic acid of the first component.

[0280] In some embodiments, a sequence of the nucleic acid scaffold strand may be suitable for hybridizing to a sequence of a nucleic acid of the first component.

[0281] In some embodiments, a sequence of a nucleic acid staple strand may be suitable for hybridizing to a sequence of a nucleic acid staple strand of the first component. [0282] The nucleic acid origami module may comprise a sleeve and an extended element. The sleeve and the extended element each comprises a longitudinal axe. The extended element is disposed concentrically within the sleeve, with the longitudinal axe of the extended element being parallel to the longitudinal axe of the sleeve. The extended element and the sleeve are movable one relative to the other.

[0283] A gap exists between the sleeve and the extended member so the extended member may freely move within the sleeve, in a direction parallel to the longitudinal axe of the sleeve and of the extended member.

[0284] The sleeve comprises a transversal dimension perpendicular to the longitudinal axe of the sleeve.

[0285] The extended element comprises a first and a second end. The first and the second ends may each be connected with the sleeve with at least one nucleic acid connector strand.

[0286] One end of the extended element may comprise a head. The head of the extended element comprises transversal dimension, perpendicular to the longitudinal axe of the extended element. The transversal dimension of the head of the extended element may be larger than the transversal dimension of the sleeve. The head of the extended element may prevent the end comprising the head from entering into the sleeve.

[0287] The extended element of the nucleic acid origami module may be arranged to have its longitudinal axe parallel, or substantially parallel, to the longitudinal axe of the extended members of the nucleic acid origami support.

[0288] The nucleic acid origami module and the nucleic acid origami support may be arranged together for having the longitudinal axe of the extended element the nucleic acid origami module perpendicular or substantially perpendicular to the surface of interest, when the nucleic acid origami modular device is positioned at the surface of interest.

[0289] An end of the extended element may be proximal to the surface of interest and the other end may be distal from the surface of interest.

[0290] The distal end may comprise a head as above indicated. The head of the extended element may be arranged to be on a distal position relative to the surface of interest.

[0291] The end of the extended member distal to the surface of interest may be functionalized with at least one binding moiety able to bind a complementary moiety present at the surface of interest. [0292] The proximal end may be functionalized with one to six binding moieties.

[0293] The proximal end may be functionalized with at least three binding moieties or may be functionalized with three binding moieties.

[0294] The binding moiety(ies) may covalently or non-covalently be attached to a sequence of the nucleic acid scaffold strand.

[0295] In some embodiments, one or a plurality of binding moiety(ies) may covalently or non-covalently be attached to one nucleic acid scaffold strand.

[0296] In some embodiments, one binding moiety may covalently or non-covalently be attached to one nucleic acid scaffold strand.

[0297] The binding moiety(ies) may covalently or non-covalently be attached to a nucleic acid staple strand hybridizing the nucleic acid scaffold. It is usually more practical to have the binding moiety(ies) covalently or non-covalently attached to a nucleic acid staple strand as it allows designing in efficient and cost-effective ways various nucleic acid modular devices of the disclosure of various specificities and affinities for different surfaces of interest or different regions of a same surface of interest.

[0298] The moiety(ies) may covalently be attached to a nucleic acid staple strand.

[0299] In some embodiment, a binding moiety may be an oligonucleotide having a part of its sequence complementary to a sequence of a nucleic acid of the extended element and a part of its sequence complementary to a sequence of nucleic acid present at the surface of interest.

[0300] A binding moiety may be an aptamer.

[0301] As used herein, an “aptamer” refers to a nucleic acid molecule, such as RNA or DNA, that is capable of binding to molecules.

[0302] A binding moiety may be an antibody or an antigen-binding fragment thereof, or a ligand capable of binding to a complementary moiety present at the surface of interest.

[0303] A complementary moiety may be a sub-unit, a domain, a motif and/or an epitope of a molecule present at the surface of interest, in particular chosen among a nucleic acid sequence; a G protein-coupled receptor, such as an adrenergic receptor or an olfactory receptor; a tyrosine kinase receptor, such as an epidermal growth factor receptor, an insulin receptor, a fibroblast growth factor receptor; a mechanoreceptor, such as an integrin; a channel receptor, such as a GABA receptor; an immune receptor, such as a Toll-like receptor or a T cell receptor; or a combination thereof. [0304] The binding moiety may be an RGD-containing peptide.

[0305] An RGD-containing peptide may be chosen among fibronectin, laminin, collagen, vitronectin, and a combination thereof.

[0306] The RGD-containing peptide may be a cyclic RGD-oligonucleotide.

[0307] A sequence of a nucleic acid strand to be functionalized with a binding moiety may comprise or consist in a sequence having at least about 70% to about 100% identity with a sequence selected from SEQ ID NOs: 156 to 158.

[0308] A sequence of a nucleic acid strand to be functionalized with a binding moiety may comprise or consist in a sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% identity with a sequence selected from SEQ ID NOs: 156 to 158.

[0309] A sequence of a nucleic acid strand to be functionalized with a binding moiety may consist in a nucleic acid sequence selected from SEQ ID NOs: 156 to 158.

[0310] The sleeve comprises an internal side and an external side. The internal side is facing the extended element of the nucleic acid origami module inserted in the sleeve. The external side comprising a site of attachment of the nucleic acid module to the head of the nucleic acid support.

[0311] At least one nucleic acid connector strand may connect one end of the extended element and the sleeve.

[0312] At least one nucleic acid connector strand may connect the distal end of the extended element and the sleeve.

[0313] At least one nucleic acid connector strand may connect the head of the end of the extended element and the sleeve.

[0314] Both ends of the extended element may each be connected to the sleeve with at least one nucleic acid connector strand.

[0315] Two to eighteen nucleic acid connector strands may connect the sleeve with an end or both end of the extended element.

[0316] In some embodiments, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or eighteen strands of nucleic acid connector strands may connect the sleeve with an end or both ends of the extended element.

[0317] In some embodiments, six nucleic acid connector strands may connect the sleeve with an end of the extended element.

[0318] Six nucleic acid connector strands may connect the sleeve with the distal end of the extended element.

[0319] Six nucleic acid connector strands may connect the sleeve with the head of the extended element.

[0320] Six nucleic acid connector strands may connect the sleeve with both ends of the extended element.

[0321] A nucleic acid connector strand may be single-stranded.

[0322] A nucleic acid connector strand may be double-stranded.

[0323] In some embodiments, a nucleic acid connector strand is single-stranded.

[0324] A nucleic acid connector strand may be of a length from about 10 nucleotides (nt) to about 200 nt, or about 50 nt to about 150 nt, or about 70 nt to about 1 10 nt.

[0325] A nucleic acid connector strand may be of a length of at least about 10 nt or more, 15 nt or more, 20 nt or more, 25 nt or more, 30 nt or more, 35 nt or more, 40 nt or more, 45 nt or more, 50 nt or more, 55 nt or more, 60 nt or more, 65 nt or more, 70 nt or more, 75 nt or more, 80 nt or more, 85 nt or more, 90 nt or more, 95 nt or more, 97 nt or more, 100 nt or more, 105 nt or more, 110 nt or more, 1 15 nt or more, 120 nt or more, 125 nt or more, 130 nt or more, 135 nt or more, 140 nt or more, 145 nt or more, 150 nt or more, 155 nt or more, 160 nt or more, 165 nt or more, 170 nt or more, 175 nt or more, 180 nt or more, 185 nt or more, 190 nt or more, 195 nt or more, or about 200 nt or more.

[0326] A nucleic acid connector strand may be of a length from about 90 nt to 105 nt.

[0327] A nucleic acid connector strand may be of a length of about 90 nt, 91 nt, 92 nt, 93 nt, 94 nt, 95 nt, 96 nt, 97 nt, 98nt, 99 nt, 100 nt, 101 nt, 102 nt, 103 nt, 104 nt, or 105 nt.

[0328] In some embodiments, a nucleic acid connector strand may be of a length of about 97 nt.

[0329] In some embodiments, a nucleic acid connector strand may be of a length of about 60 nt. [0330] In some embodiments, a nucleic acid connector strand may be of a length of about 30 nt.

[0331] Nucleic acid staple strands which may be used to obtain a nucleic acid origami modular device of the disclosure with nucleic acid connector strands of a length of about 97 nt may comprise or consist in nucleic acids having at least about 70% to about 100% sequence identity with a sequence selected from SEQ ID NOs: 131 , 68, 77, 76, 129, 60, 1 12, 84, 86 and 138.

[0332] Nucleic acid staple strands which may be used to obtain a nucleic acid origami modular device of the disclosure with nucleic acid connector strands of a length of about 97 nt may comprise or consist in nucleic acids having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with a sequence selected from SEQ ID NOs: 131 , 68, 77, 76, 129, 60, 1 12, 84, 86 and 138.

[0333] Nucleic acid staple strands which may be used to obtain a nucleic acid origami modular device of the disclosure with nucleic acid connector strands of a length of about 60 nt may comprise or consist in nucleic acids having at least about 70% to about 100% sequence identity with a sequence selected from SEQ ID NOs: 388 to 397.

[0334] Nucleic acid staple strands which may be used to obtain a nucleic acid origami modular device of the disclosure with nucleic acid connector strands of a length of about 60 nt may comprise or consist in nucleic acids having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with a sequence selected from SEQ ID NOs: 388 to 397.

[0335] Nucleic acid staple strands which may be used to obtain a nucleic acid origami modular device of the disclosure with nucleic acid connector strands of a length of about 30 nt may comprise or consist in nucleic acids having at least about 70% to about 100% sequence identity with a sequence selected from SEQ ID NOs: 398 to 407.

[0336] Nucleic acid staple strands which may be used to obtain a nucleic acid origami modular device of the disclosure with nucleic acid connector strands of a length of about 30 nt may comprise or consist in nucleic acids having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with a sequence selected from SEQ ID NOs: 398 to 407. [0337] The nucleic acid connector strands connecting one end of the extended element with the sleeve may have all the same length.

[0338] A nucleic acid connector strand connected to the sleeve and the end of the extended element distal from the surface of interest may have a length of 97 nt.

[0339] A nucleic acid connector strand connected to the sleeve and the end of the extended element distal from the surface of interest may be an entropic spring. The entropic spring may exert a force moving back and forth the extended element within the sleeve in a direction parallel to the longitudinal axes of the extended element and of the sleeve.

[0340] A portion of the least one nucleic acid connector strand connected to the sleeve and the end of the extended element distal from the surface of interest may be folded on said distal end.

[0341] The folding of a portion of a nucleic acid connector strand may be assured with staple strands. The folding of a portion of a nucleic acid connector may allow controlling the length of the nucleic acid connector strand and therefore the tension exert by the nucleic acid connector strand on the extended element, which is movable within the sleeve, the sleeve being attached to the first component, and the first component attaching the device on a surface of interest.

[0342] A portion of the least one nucleic acid connector strand connected to the sleeve and the end of the extended element distal from the surface of interest may be folded on said head of said distal end.

[0343] A portion of the least one nucleic acid connector strand connected to the sleeve and the end of the extended element distal from the surface of interest may be folded in a form of a loop, preferably in a form of a plurality of continued loops.

[0344] In some embodiments, a portion of the nucleic acid connector strands may be folded on the head of the extended element of the nucleic acid origami module.

[0345] In some embodiments, a portion of the nucleic acid connector strands may be folded on the head of the extended element in a form of a loop.

[0346] In some embodiments, a portion of the nucleic acid connector strands may be folded on the head of the extended element in a form of a plurality of continued loop.

[0347] In some embodiments, the portion of a nucleic acid connector strand folded on the head of the extended element may be of a length from about 0 nt to about 100 nt.

[0348] In some embodiments, the portion of a nucleic acid connector strand folded on the head of the extended element may be of a length of about 0, 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, 31 , 32, 33,

34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57,

58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69,70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 ,

82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or about 100 nt.

[0349] By defining the number of nucleic acid connector strands connecting the sleeve to the end of the extended element distal from the surface of interest, or the head of the extended element, and by defining its length, it is possible to adjust at the pN unit level the force exerted by the nucleic acid module onto the surface of interest.

[0350] Nucleic acid connector strands connecting the sleeve to the end of the extended element distal from the surface of interest, or the head of the extended element, may also be used to remotely activate a nucleic acid origami modular device of the disclosure.

[0351] For example, the use of nucleic acid sequence, e.g., single-stranded oligonucleotide, complementary to the sequences of the nucleic acid connector strands allows maintaining the nucleic acid connector strands in an extended position and therefore the distal end of the extended element at distance from the surface of interest.

[0352] In case where the functionalized distal end of the extended member has bound a complementary moiety at the surface of interest, then it is possible, by adding the complementary oligonucleotide, to force the extended element to draw on this complementary moiety in a stretched position.

[0353] The use of degradable complementary oligonucleotides allows the relaxation of the nucleic acid connector strands, and therefore the relaxation of the stretched complementary moiety. Degradable complementary oligonucleotides may be UV degradable azobenzene oligonucleotide.

[0354] As indicated, a nucleic acid origami module comprises at least one nucleic acid scaffold strand and a plurality of nucleic acid staple strands. The staple strands are designed to hybridize to the scaffold strand to fold it into a nucleic acid origami module.

[0355] The strands are single-strand nucleic acids.

[0356] In some embodiments, a nucleic acid origami module may comprise one nucleic acid scaffold strand.

[0357] In some embodiments, a nucleic acid origami module may comprise a plurality of nucleic acid scaffold strands folded into the different parts of the module: sleeve, extended element and nucleic acid connector strand(s), the different parts being held together in a nucleic acid origami module by hybridization.

[0358] In some embodiments, a nucleic acid origami module may comprise a plurality of nucleic acid scaffold strands each joined together to form a sleeve, an extended member, and nucleic acid connector strand(s).

[0359] In some embodiments, a nucleic acid origami module may comprise a single nucleic acid scaffold strand folded into the module with a plurality of nucleic acid staple strands.

[0360] A scaffold strand may be of a length of about 15 to about 000 nucleotides (nt).

[0361] A scaffold strand may be of a length of about 50 to about 19 000 nucleotides (nt), or about 100 to about 18 500 nt, or about 1 000 to about 18 000 nt, or about 1 500 to about 17 000 nt, or about 2 000 to about 16 000 nt, or about 3 000 to about 15 000 nt, or about 4 000 to about 14 000 nt, or about 4 500 to about 13 000 nt, or about 5 000 to about 12 000 nt, about 5 500 to about 1 1 000 nt, or about 6 000 to about 10 000 nt, or about 6 500 to about 9 000 nt, about 6 800 to about 8 500 nt, or about 7 000 to about 8 400 nt, or about 7 200 to about 8 200 nt, or about 7 400 to about 8 100 nt, or about 7 450 to about 8 000 nt, or about 7 500 to about 7 800 nt, or about 7 500 to about 7 600 nt.

[0362] A single scaffold strand may be of a length of about 7 560 nt.

[0363] In some embodiments, a sequence of the nucleic acid scaffold strand of a nucleic acid origami module may comprise or consist in a sequence having at least about 70% to about 100% identity with the sequence SEQ ID NOs: 408.

[0364] A sequence of the nucleic acid scaffold strand of an origami module may comprise or consist in a sequence having at least about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with the sequence SEQ ID NOs: 408.

[0365] A sequence of the nucleic acid scaffold strand of an origami module may consist in the nucleic acid sequence SEQ ID NOs: 408.

[0366] A staple strand for an origami module may be a nucleic acid of a length of about 12 to about 100 nt.

[0367] A staple strand may be a nucleic acid of a length of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64,

65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88,

89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or about 100 nt.

[0368] A staple strand may be a nucleic acid of a length of about 10 nt to about 100 nt.

[0369] A staple strand may be a nucleic acid of a length of about 20 nt to about 70 nt.

[0370] The number of staple strands to form a nucleic acid origami module may be determined by considering the number of nucleotides in a nucleic acid scaffold strand and the number of nucleotides in the smallest nucleic acid staple strand. Specifically, the maximum number of staple strands to be used in a nucleic acid origami of the disclosure is the ratio of the number of nucleotides in a nucleic acid scaffold strand to the number of nucleotides in the smallest nucleic acid staple strand.

[0371] The number of staple strands to form a nucleic acid origami module may range from about 10 to about 1000 staple strands.

[0372] The number of staple strands to form a nucleic acid origami support may range from about 20 to about 700 staple strands, or from about 50 to about 600 staples strands, or from about 100 to about 450, or from about 150 to about 400 staples strands.

[0373] A sequence of a nucleic acid staple strand of a sleeve for an origami module may comprise or consist in a sequence having at least about 70% to about 99% sequence identity with a sequence selected from SEQ ID NOs: 1 to 54.

[0374] A sequence of a nucleic acid staple strand of a sleeve for an origami module may comprise or consist in a sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with a sequence selected from SEQ ID NOs: 1 to 54.

[0375] A sequence of a nucleic acid staple strand of a sleeve for an origami module may consist in a sequence as set forth in SEQ ID NOs: 1 to 54.

[0376] A sequence of a nucleic acid staple strand of an extended element for an origami module may comprise or consist in a sequence having at least about 70% to about 99% sequence identity with a sequence selected from SEQ ID NOs: 55 to 139 and 388 to 407. [0377] A sequence of a nucleic acid staple strand of an extended element for an origami module may comprise or consist in a sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with a sequence selected from SEQ ID NOs: 55 to 139 and 388 to 407.

[0378] A sequence of a nucleic acid staple strand of an extended element for an origami module may consist in a sequence as set forth in SEQ ID NOs: 55 to 139 and 388 to 407.

[0379] As indicated, a nucleic acid origami module comprises at least one nucleic acid scaffold strand and a plurality of nucleic acid staple strands. The staple strands are designed to hybridize to the scaffold strand to fold it into a nucleic acid origami module.

[0380] The strands are single-strand nucleic acids.

[0381] In some embodiments, a nucleic acid origami module may comprise one nucleic acid scaffold strand.

[0382] In some embodiments, a nucleic acid origami module may comprise a plurality of nucleic acid scaffold strands folded into the different parts of the support: sleeve, extended element and nucleic acid connector strand.

[0383] In some embodiments, a nucleic acid origami support may comprise a single nucleic acid scaffold strand folded into the module with a plurality of nucleic acid staple strands.

[0384] A nucleic acid origami module may comprise a sleeve and an extended element. The extended element comprises a first and a second end. The first and the second ends may each be connected with the sleeve with at least one nucleic acid connector strand. One end of the extended element, the end configured for being distal from the surface of interest, may comprise a head.

[0385] The extended element may comprise at least one helix or more.

[0386] The extended element may comprise at least two helix or more.

[0387] The extended element may comprise of about one to twelve helix/ces.

[0388] The extended element may comprise one, two, tree, four, five, six, seven, eight, nine, ten, eleven or twelve helix/ces.

[0389] The extended element may comprise six helices of nucleic acid. [0390] The extended element may comprise at least one bundle of six-helix bundle or more.

[0391] The extended element may comprise of about one to six bundles of six-helix bundle.

[0392] The extended element may comprise one bundle of six-helix bundle.

[0393] The six-helix bundle may have a tube-like shape.

[0394] The extended element may be of a length ranging from about 20 nm to about 100 nm or more.

[0395] The extended element may be of a length of about 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, 50 nm or more, 55 nm or more, 60 nm or more, 65 nm or more, 70 nm or more, 75 nm or more, 80 nm or more, 85 nm or more, 90 nm or more, 95 nm or more, or of about 100 nm or more.

[0396] The extended element may be of a length from about 40 nm to 90 nm, or about 50 nm to 85 nm, or about 60 to 90 nm.

[0397] The extended element may be of a length of about 80 nm.

[0398] The head of the extended element may comprise at least eighteen helices.

[0399] The head of the extended element may comprise about eighteen to fifty helices.

[0400] The head of the extended element may comprise about 18, 19, 20, 21 , 22,

23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46,

47, 48, 49 or 50 helices.

[0401] The head of the extended element may comprise thirty-six helices.

[0402] The head of the extended element may comprise at least six bundles of six- helix bundle (6-HB).

[0403] The head of the extended element may comprise six to twelve bundles of six-helix bundle.

[0404] The head of the extended element may comprise six, seven, eight, nine, ten, eleven or twelve bundles of six-helix bundle.

[0405] The head of the extended element may comprise six bundles of six-helix bundle (6-HB). [0406] The head of the extended element may have a length from about 5 nanometers (nm) to 40 nm.

[0407] The head of the extended element may have a length of about 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm 35 nm, 36 nm, 37 nm, 38 nm, 39, or of about 40 nm.

[0408] The head of the extended element may have a length of about 20 nm.

[0409] A gap is present between the sleeve and the extended member.

[0410] The gap may be of a length from about 0.1 nm to 3 nm.

[0411] The gap may be of a length of about 0.1 nm, 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 1.1 nm, 1 .2 nm, 1 .3 nm, 1 .4 nm, 1 .5 nm, 1 .6 nm, 1 .7 nm, 1.8 nm, 1.9 nm, 2 nm, 2.1 nm, 2.2 nm, 2.3 nm, 2.4 nm, 2.5 nm, 2.6 nm, 2.7 nm, 2.8 nm, 2.9 nm, or of about 3 nm or more.

[0412] The gap may be of a length of about 2 nm.

[0413] The sleeve may have a cylinder-like shape.

[0414] The sleeve of the nucleic acid origami module may comprise at least eighteen helices.

[0415] The sleeve of the nucleic acid origami module may comprise about eighteen to fifty helices.

[0416] The sleeve of the nucleic acid origami module may comprise 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 helices.

[0417] The sleeve of the nucleic acid origami module may comprise 32 to 50 helices.

[0418] The sleeve of the nucleic acid origami module may comprise thirty-two helices.

[0419] The sleeve may comprise at least six bundles of six-helix bundle (6-HB).

[0420] The sleeve may comprise six to twelve bundles of six-helix bundle.

[0421] The sleeve may comprise six, seven, eight, nine, ten, eleven or twelve bundles of six-helix bundle.

[0422] The sleeve may comprise six bundles of six-helix bundle.

[0423] The sleeve may be of a length ranging from about 5 nm to about 50 nm or more. [0424] The sleeve may be of a length of about 5 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, 50 nm or more,

[0425] The sleeve may be of a length from about 25 to 35 nm.

[0426] The sleeve may be of a length of about 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 or 35 nm.

[0427] The sleeve may be of a length of about 30 nm.

[0428] In some embodiments, the sleeve may comprise at least one sequence of nucleic acid complementary to a sequence of the nucleic acid origami support.

[0429] The sleeve may comprise at least one sequence of nucleic acid complementary to a sequence of the head of a nucleic acid origami support.

[0430] The sleeve may comprise from one to eight sequences of nucleic acid(s) complementary to one to eight sequences of the nucleic acid origami support.

[0431] In some embodiments, the sleeve of the nucleic acid origami module may comprise eight sequences of nucleic acids complementary to eight sequences of the nucleic acid origami support.

[0432] In some embodiments, the sleeve of the nucleic acid origami module may comprise sixteen sequences of nucleic acids complementary to eight sequences of a first nucleic acid origami support and to eight sequences of a second nucleic acid origami support.

[0433] The sleeve of the nucleic acid origami module may be arranged to have the sequences of nucleic acids complementary to sequences of the nucleic acid origami support at close proximity from one to each other.

[0434] A sequence of a nucleic acid complementary to a sequence of the nucleic acid origami support may comprise or consist in a sequence having at least about 70% to about 100% identity with a sequence selected from SEQ ID NOs: 140 to 155.

[0435] A sequence of a nucleic acid complementary to a sequence of the nucleic acid origami support may comprise or consist in a sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% identity with a sequence selected from SEQ ID NOs: 140 to 155. [0436] A sequence of a nucleic acid complementary to a sequence of the nucleic acid origami support may consist in a sequence selected from SEQ ID NOs: 140 to 155.

Assembly of the first and second component

[0437] Once the first and second components of a nucleic acid modular device of the disclosure assembled together, the obtained device may have a dimension of length x depth x height nm of about 90x90x60 nm to 150x150x120 nm in size.

[0438] In some embodiments, a nucleic acid origami modular device may have a dimension of length x depth x height nm of about 90x90x60 nm to about 150x150x120 nm in size, or about 95x95x65 nm to about 145x145x115 nm, or about 100x100x70 nm to about 140x140x1 10 nm, or about 105x105x75 nm to about 135x135x105 nm, or about 100x100x80 nm to about 130x130x100 nm, or about 100x100x80 nm to about 120x120x95 nm.

[0439] In some preferred embodiments, the nucleic acid origami modular device may have a dimension of about 1 14x114x90 nm in size.

[0440] In some embodiments, a nucleic acid origami modular device of the disclosure, may comprise:

[0441] a first module comprising (a) two nucleic acid origami supports, and

[0442] a second module comprising (b) a nucleic acid origami module,

[0443] each of the two nucleic acid origami supports being attached to the nucleic acid origami module,

[0444] wherein

[0445] the nucleic acid origami module comprises:

[0446] a sleeve, and

[0447] an extended element,

[0448] the sleeve and the extended element having each a longitudinal axe, with the longitudinal axe of the extended element being parallel to the longitudinal axe of the sleeve,

[0449] the extended being disposed concentrically within the sleeve,

[0450] said extended element and said sleeve being movable one relative to the other in a direction of the longitudinal axe of the extended member, and [0451] the two nucleic acid origami supports being attached to said sleeve, wherein

[0452] each of the two nucleic acid origami supports comprising:

[0453] a head for attaching the nucleic acid origami support to the nucleic acid origami module,

[0454] at least one extended member projecting from said head, said each extended members being connected by one end to said head and by the other end to at least one base, said base comprising said at least one anchor element.

[0455] The nucleic acid origami module and the nucleic acid origami supports may be as above described.

[0456] The sleeve may be comprised of six bundles, each being a six-helix bundle.

[0457] The extended element may be comprised of a six-helix bundle.

[0458] The extended element comprises a first and a second end. The first and the second ends may each be connected with the sleeve with at least one nucleic acid connector strand. The first and the second ends may each be connected with the sleeve with at least six nucleic acid connector strands.

[0459] An end of the extended element maybe arranged for being proximal to a surface of interest and the other end may arranged for being distal from the surface of interest.

[0460] The distal end may comprise configured for preventing the distal end of the extended element from entering into the sleeve.

[0461] The head may be comprised of six bundles, each having a six-helix bundle.

[0462] The sleeve and the extended element are arranged one relative to the other for maintaining a gap between said sleeve and said extended element

[0463] The end of the extended element being arranged for being proximal to a surface of interest may be functionalized with at least one moiety able to bind a complementary moiety present at the surface of interest.

[0464] The head of a nucleic acid origami support may be comprised of eight bundles, each being a six-helix bundle. The head may attach the nucleic acid origami support to the nucleic acid origami module by means of at least one sequence of nucleic acid of the head complementary to at least one sequence of nucleic acid of the sleeve of the nucleic acid origami module. [0465] The extended member of the nucleic acid origami support may be comprised of two bundles, each being a six-helix bundle.

[0466] The base may comprise at least one anchor member.

[0467] An anchor member may a nucleic acid staple strand functionalized with at least one moiety suitable to adhere to or insert in said surface of interest.

[0468] A suitable moiety may be a cholesterol.

[0469] Each extended member and its base may further be connected by at least one nucleic acid connector strand. The nucleic acid connector strand may be a two doublestranded strand.

Methods for preparing a nucleic acid origami modular device and kits

[0470] A nucleic acid origami modular device as disclosed herein may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic, or computer tool, either alone or in combination.

[0471] A nucleic acid origami modular device as disclosed herein may be designed using caDNAno (https://cadnano.org/), which is a computer-aided design tool for nucleic acid origami synthesis.

[0472] The modules of the nucleic acid origami modular device may be obtained according to any known methods in the nucleic acid origami field.

[0473] The ratio of scaffold strand per staple strand may be from about 1 :1 to about 1 :100.

[0474] In some embodiments, the ratio of scaffold strands per staple strands may be from about 1 :2 to about 1 :50, or from about 1 :4 to about 1 :25, or from about 1 :8 to about 1 :16.

[0475] In some embodiments, the ratio of scaffold strand per staple strand may be about 1 :10.

[0476] The nucleic acid origami modules and supports may be obtained by thermal annealing suitable for the folding of a scaffold strand into a module or a support with one or more staples stand(s). Suitable thermal annealing conditions may be, for example, ramp: 65°C for 15 minutes, 60°C to 40°C -1 °C per every 2 hours, then held at 10°C . [0477] The obtained nucleic acid origami modules and supports may be purified by any known method in the art, such as size-exclusion chromatography, gel electrophoresis, PEG-precipitation, Rate-Zonal Centrifugation or affinity chromatography.

[0478] The present disclosure also relates to a method for preparing a nucleic acid origami modular device as described herein.

[0479] The method may comprise at least a step of mixing a first composition comprising nucleic acid origami supports according to the disclosure and a second composition comprising nucleic acid origami modules according to the disclosure.

[0480] The ratio of the first composition to the second composition is adapted according to the number of nucleic acid origami supports attached to the nucleic acid origami module.

[0481] The ratio of the first composition to the second composition may be range from 1 :1 to 4:1 and may be 2:1 .

[0482] In some embodiments, to ensure having the intended ratio of nucleic acid supports per nucleic acid origami module, it is possible to work with slight excess of supports compared to the theoretical ratio. For example, to reach a ratio of 2 supports per module, one may work at a ratio of 2.1 :1 , 2.2:1 , 2.3:1 , or 2.4:1 .

[0483] In some embodiments, the ratio of the first composition to the second composition may be 2.2:1 .

[0484] The first and second compositions may comprise a buffer.

[0485] A suitable buffer may comprise a salt for ensuring the ionic force necessary for the assembly of the first component with the second component, and for the folded of the origamis to be maintained.

[0486] A buffer may be chosen among TAPS, Bisine, Tris, Tris-HCI, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylat or MES.

[0487] In some embodiments, a buffer may be a Tris-HCI buffer.

[0488] A salt suitable for a composition to be used in a method of the disclosure may be NaCI, KCI, and MgCh

[0489] A salt may be used at a concentration ranging from about 5 mM to about 500 mM.

[0490] A suitable salt may be MgCh. [0491] MgCh may be present in a concentration ranging from about 5 mM to about 40 mM, or from about 11 mM to about 35 mM and for example may be about 18 mM.

[0492] In some embodiments, MgCls may be about 5 mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11 mM, 12 mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21 mM, 21 mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 31 mM, 32mM, 33mM, 34mM, 35mM, 36mM, 37mM, 38mM, 39mM, or 40 mM.

[0493] In some embodiments, MgCls may be about 20 mM or about 18 mM.

[0494] In some embodiments, the first and second compositions may have a pH ranging from about of 7 to 9.

[0495] The pH may be about 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.

[0496] In some embodiments, the first and the second compositions may have a pH of about 8.

[0497] After mixing of the first and second compositions, the first and second components of the nucleic acid modular device of the disclosure are left incubated at a temperature and for a time suitable for the assembly of the first and second components in a nucleic acid origami modular device of the disclosure.

[0498] In some embodiments, a suitable time may range from about 10 minutes to about 168 hours, or from about 30 minutes to about 120 hours, or from about 1 hour to about 100 hours, or from about 2 hours to about 80 hours, or from about 5 hours to about 60 hours, or from about 10 hours to about 50 hours or from about 15 hours to about 30 hours.

[0499] In some embodiment, a suitable time may be about 20 hours.

[0500] In some embodiments, a suitable temperature may range from about 10°C to about 45°C, or from about 15°C to about 42°C, or from about 20°C to about 40°C, or from about 25°C to about 39°C, or from about 30°C to about 38°C.

[0501] In some embodiment, a suitable temperature may be about 37°C.

[0502] For example, suitable time and temperature may be 20h at 37°C.

[0503] A nucleic acid origami modular device may be purified with any method known in the art, such as size-exclusion chromatography, gel electrophoresis, PEG- precipitation, Rate-Zonal Centrifugation, or affinity chromatography. [0504] In some embodiments, a method for preparing a nucleic acid origami modular device as described further comprises a step of incubating the purified nucleic acid origami modular device with a ligand-conjugated oligonucleotide as described in the present disclosure.

[0505] In some embodiments, a suitable incubation may be performed at a temperature and for a time suitable to attach the ligand-conjugated oligonucleotide to a complementary sequence of the nucleic acid scaffold strand of the module.

[0506] In some embodiment, a suitable temperature may be about 37°C.

[0507] In some embodiment, a suitable time of incubation may be at least about 30 minutes.

[0508] For example, a suitable time and temperature to attach the ligand-conjugated oligonucleotide to a complementary sequence of the nucleic acid scaffold strand of the module may be 37°C for at least 30 minutes.

[0509] The disclosure also relates to a kit for preparing a nucleic acid origami modular device as disclosed herein. The kit may comprise a first container comprising a first composition comprising nucleic acid origami supports as disclosed herein and a second container comprising a second composition comprising nucleic acid origami modules as disclosed herein.

[0510] The first and second compositions may be as detailed above.

[0511] A kit may further comprise an instruction indicating to mix a first and a second composition as disclosed.

[0512] The first composition of the kit may comprise a concentration of the second component suitable to achieve, when mixed with a first composition, one of the ratios aboveindicated.

[0513] For example, for a nucleic acid origami modular device comprising two nucleic acid origami supports and one nucleic acid origami module, the first composition containing the supports may comprise at least 2, or 2.1 , or 2.2, or 2.3, or 2.4, and for example 2.2, -fold molar excess of nucleic acid origami supports compared to the second composition containing the nucleic acid origami module.

[0514] The kit may further comprise an instruction indicating to adjust the amount of the first composition relative to the amount of the second composition according to the ratio of the nucleic acid origami support to the nucleic acid module to achieve. [0515] A kit may further comprise means and instructions for purifying the obtained nucleic acid origami modular device.

Uses and methods

[0516] A nucleic acid origami modular device may be used for exerting and/or measuring a force at a surface of interest.

[0517] The disclosure relates to a use of a nucleic acid origami modular device disclosed herein for exerting a force on the surface of interest, optionally said force inducing a mechanical constraint.

[0518] The force may be in the range of piconewton (pN). The force may range from about 0.5 to about 100 pN.

[0519] The force may be in the range of about 0.5 pN to about 90 pN, or about 0.7 to about 85 pN, or about 1 .0 to about 83 pN, or about 1 .7 pN to about 80 pN.

[0520] After placing a nucleic acid origami modular device of the disclosure at a surface of interest, the first and the second components can cooperate together for exerting a force on the surface of interest.

[0521] The first component which is/are a nucleic acid origami support/s with anchor members allow/s attaching the nucleic acid origami modular device at a surface of interest.

[0522] The second component, i.e., the nucleic acid origami module, is an actuator. That is, the nucleic acid origami module is configured to move relative to the nucleic acid origami support and to interact with the surface of interest.

[0523] In case of a module, as previously detailed, comprising a sleeve and an extended element, the extended element may move within the sleeve. Thanks to the support/s attached to the sleeve, the movement of the extended element, parallel to its longitudinal axe, may be perpendicular to the surface of interest. With functionalization of the end of the extended element proximal from the surface of interest, interactions between the nucleic acid origami modular device is possible. An interaction can be, for example, the binding of the ligand/s functionalizing the proximal end to a mechanoreceptor.

[0524] Both ends of the extended element are connected to the sleeve with nucleic acid connector strands. By defining the number of nucleic acid connector strands connecting the sleeve to the end of the extended element distal from the surface of interest, or the head of the extended element, and by defining their lengths, it is possible to adjust at the pN unit level the force exerted by the nucleic acid module onto the surface of interest. [0525] The nucleic acid connector strands act as entropic springs allowing the extended element to move back and forth within the sleeve, in a direction parallel to the longitudinal axes of the extended element and of the sleeve, and perpendicular to a surface of interest.

[0526] The nucleic acid origami support/s are attached to the surface of interest. Therefore, it/they may oppose to the nucleic acid origami module movement, e.g., the movement of the extended element, so that the movement of the nucleic acid origami module is translated into a force exerted at the surface of interest.

[0527] The exerted force may induce a mechanical constraint at the surface of interest, such as a pression, a traction, a twist, or a stretch.

[0528] The mechanical constraint may be used for activating a receptor on a surface of interest. For example, in case of an interaction with a mechanoreceptor, the extended element, moving back and forth within the sleeve, may draw (the force is translated into traction) on the mechanoreceptor and induce its activation.

[0529] The mechanical constraint may be used for drilling a surface of interest.

[0530] The mechanical constraint may be used for opening a channel on a surface of interest.

[0531] The mechanical constraint may be used for removing a molecule on a surface of interest.

[0532] The nucleic acid connector strands connecting the sleeve to the end of the extended element distal from the surface of interest, or the head of the extended element, may also be used to remotely activate a nucleic acid origami modular device of the disclosure.

[0533] Nucleic acid sequence, e.g., single-stranded oligonucleotide, complementary to the sequences of the nucleic acid connector strands may bind to the nucleic acid connector strands and maintain them in extension. Therefore, the distal end of the extended element is maintained away from the surface of interest.

[0534] In case where the functionalized distal end of the extended member has bound a complementary moiety at the surface of interest, then it is possible, by adding the complementary oligonucleotide, to force the extended element to draw on this complementary moiety in a stretched position.

[0535] The use of degradable complementary oligonucleotides allows the relaxation of the nucleic acid connector strands, and therefore the relaxation of the stretched complementary moiety. Degradable complementary oligonucleotides may be UV degradable azobenzene oligonucleotide.

[0536] The disclosure relates to a method for exerting a force, optionally for inducing a mechanical constraint, on a surface of interest, the method comprising at least a step of contacting a nucleic acid origami modular device of the disclosure with said surface, in conditions suitable for the nucleic acid origami support to attach to said nucleic acid origami modular device to said surface of interest, and for the nucleic acid module to interact with said surface of interest, wherein said interaction exerts a force on said surface of interest.

[0537] The method may further comprise a step of adding nucleic acid sequence, e.g., single-stranded oligonucleotide, complementary to the sequences of the nucleic acid connector strands connecting the sleeve and the distal end, or the head, of the extended member.

[0538] The oligonucleotides may be degradable. The oligonucleotide may be UV degradable azobenzene oligonucleotide.

[0539] A surface of interest may be a cell membrane.

[0540] In some embodiments, a cell membrane of interest may be from a cell chosen from an erythrocyte, a platelet, a bone marrow cell, an endothelial cell, a lymphocyte, a hepatocyte, a neuron, a bronchial endothelial cell, an epidermal cell, a respiratory interstitial cell, an adipocyte, a dermal fibroblast or a muscle cell.

[0541] In some embodiments, a cell membrane of interest may be a cell membrane of a cancer cell.

[0542] Since the nucleic acid origami modular device can attached to a surface of a cell and need not to be maintained elsewhere to exert a force on the surface, then the device may be use on surface of cells in suspension.

[0543] Since it is possible to calibrate at the pN unit level, by choosing the number and length of nucleic acid connector strands between the distal end of the extended element and the sleeve, the intensity of the force exerted by the extended member, it is then possible to measure, by using a set of differently calibrated devices the force at which a given element present at the surface of a cell is activated. For example, in case of mechanoreceptor, such as an integrin, it is possible to measure the force at which the integrin is unfolded and activate its intracellular signal.

[0544] The following working examples illustrate the embodiments of the disclosure that are presently best known. However, it is to be understood that the following are only exemplary or illustrative of the application of the principles of the present disclosure. Numerous modifications and alternative compositions, methods, and systems may be devised by those skilled in the art without departing from the spirit and scope of the present disclosure.

[EXAMPLES]

EXAMPLE 1 : Materials & Methods

Oligonucleotides

[0545] Desalted staple oligonucleotides were purchased from either Eurofins or Integrated DNA Technologies (IDT) corresponding to SEQ ID NOs: 1 to 158 and 388 to 407 for the DNA origami piston (or DNA origami module) and of SEQ ID NOs: 159 to 371 for the DNA origami landing legs (DNA origami supports) functionalized with cholesterol. A DNA scaffold of a length of 7560 nucleotides (p7560) was purified from the M 13 bacteriophage replicated in XL1 blue strain Escherichia coli (Agilent, USA). The DNA scaffold consisting of the nucleic acid sequence as set forth in SEQ ID NO: 408. The same DNA scaffold has been used for the DNA origami module and for each DNA origami support.

Design and assembly of the nucleic acid origami modular device (Nanowinch)

[0546] DNA structures of the modular device were designed using the honeycomb lattice on caDNAno (v.2.0).

[0547] The structure of the DNA origami piston was constructed by combining a DNA scaffold p7560 at 25nM with 200nM of the corresponding staple strands in a buffer comprising 5mM Tris-HCI, pH 8.0, 1 mM EDTA and 18mM MgCh

[0548] The structure of the DNA origami support was constructed by combining a DNA scaffold p7560 at 50 nM with 200nM of the corresponding staple strands in a buffer comprising 5mM Tris-HCI, pH 8.0, 1 mM EDTA and 18mM MgCh

[0549] The origami pistons and supports were subjected to a thermal annealing ramp: 65°C for 15 minutes, 60°C to 40°C -1 °C per every 2 hours, then held at 10°C.

Agarose gel analysis

[0550] The obtained DNA origami piston and supports were purified from 1% agarose (0.5X TBE, 45mM Tris-borate, 1 mM EDTA, pH 8.3) supplemented with 11 mM of MgCls and 0.5mg-ml ^-1 Sybr SAFE. The samples were migrated on the gel for 3h with a running buffer of 0.5X TBE, 1 1 mM MgCI2, 2.85V-crrr ¹ . To recover the DNA origami pistons and supports, the bands corresponding to properly folded DNA origami pistons and supports (Figures 15 and 16) were excised and transferred to a DNA gel extraction spin column (Merck, France) centrifuged at 5,000 g for 5 min at 4°C. Assembly of the nucleic acid origami modular device (Nano-winch)

[0551] The full DNA origami piston was combined with a 2.2-fold molar excess of the DNA origami supports separately purified from agarose gel as above described. MgCh concentration was supplemented up to a final concentration of 20mM and samples were then incubated at 37°C for 10h. Complete DNA origami modular device assembly was evaluated by migration on 1% agarose and bands containing the fully assembled nucleic acid origami modular device were excised and purified as described above. Incubation of the nucleic acid origami modular device with cholesterol-conjugated oligonucleotide was performed at 37°C for at least 30 minutes.

Lipid Vesicle Preparation

[0552] Small unilamellar vesicles (SUVs) prepared from 1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC, Avanti Lipids) were solubilized in chloroform and evaporated under a nitrogen stream. Lipid film was then resuspended in a buffer containing 5mM Tris-HCI, pH=7.8, 100mM NaCI, to 1 mg/ml final concentration. Lipid suspension was then extruded through 200 nm extrusion membranes (Avanti Lipids, U.S.A), nucleic acid origami modular devices functionalized with cholesterol were allowed to incubate with 0.25mg/ml DOPC SUVs for at least 30 minutes at room temperature before visualization by electron microscopy.

Transmission Electron Microscopy

[0553] Purified DNA origami modular devices were visualized by adsorption onto glow-discharged carbon-coated grid (Quantifoil Micro tools GmbH, Germany), stained for 60 seconds with a 2% (w/vol) aqueous uranyl acetate (Merck, France) solution, and then dried with ashless filter paper (VWR, France). Observations of EM grids were carried out on a JEOL 2200FS FEG operating at 200 kV equipped with a 4k x 4k slow-scan CDD camera (Gatan Inc.). Two-dimensional class averages were computed using EMAN2 and Imaged was used to measure distributions of the different DNA origami modular devices.

Cyclic RGD-Oligonucleotide and the control RGE-Oligonucleotide Synthesis

[0554] The oligonucleotides were elongated from deoxyguanosine commercially available solid support on an ABI 394 DNA synthesizer according to standard phosphoramidite chemistry protocols (1 pmol scale). Detritylation was performed for 65 seconds using 3 % TCA in CH2CI2. For the coupling step: benzylmercaptotetrazole (0.3 M in anhydrous CH3CN) was used as the activator along with commercially available 2’- deoxyribonucleoside-O-2-cyanoethyl, N,N- diisopropylphosphoramidites (0.075 M in CH3CN, 30 s coupling time) or proparyl-diethyleneglycol phosphoramidite 11 (0.1 M in CH3CN, 60 s coupling time). The capping step was performed with acetic anhydride using commercially available solutions (Cap A: acetic anhydride:pyridine:THF 10:10:80 v/v/v, and Cap B: 10 % N-methylimidazole in THF) for 10 seconds. The oxidation step was performed with a standard, diluted iodine solution (0.1 M I2, THF:pyridine:water 90:5:5, v/v/v) for 15 seconds. Propargyl-oligonucleotide-functionalized CPG beads were introduced into a sealed vial and treated with cone. aq. ammonia (2 mL) overnight at 40 °C. The supernatant was withdrawn and evaporated. The 5'-propargyl modified oligonucleotide 3 was dissolved in water and then analyzed by UV, HPLC and MALDI-TOF MS. The amount of oligonucleotide three, determined by UV analysis at 260 nm, was 0.60 pmol (60 % yield). The HPLC purity at 260 nm was 79%. Analytical RP- HPLC retention time: 1 1.86 min on Macherey Nagel Nucleodur 100-3 C 18 ec column (length: 75 mm, ID: 4.6 mm) with a linear gradient of 1 to 25% of CH3CN in TEAAc 0.05 M pH7. MALDI-TOF MS: m/z: [M-H]-: for C217H269N87O130P21 ealed.: 6826.48; found: 6826.93. CuAAC conjugation was performed on crude of three. To 600 nmol of three in 240 pL of water was added the RGD azide 42 1200 nmol or RGE 42 4300 nmol (240 pL of a 5 mM solution in methanol, 2eq) 240 pL of 2M TEAAc and ~0.1 mg of CuO nanopowder. The mixture was sonicated for 60 sec and then heated under microwaves assistance at 65°C for 1 h. After centrifugation, the supernatant was withdrawn and saturated EDTA solution was added (500 pL). After 5 min, the solution was desalted by size exclusion chromatography (Nap10). The resulting solution was treated for 1 h with quadrapure IDA resin. The supernatant was then evaporated. The crude was purified by HPLC on a Macherey Nagel Nucleodur C18 HTec column (length: 250 mm, ID: 10 mm), using a linear gradient from 4 to 17 % of CH3CN in 50 mM TEAAc pH 7 for 20 min, affording 200 nmol of five (33 % yield for RGD and 36 % yield for RGE). Analytical RP-HPLC retention time for RGD: 13.49 min. MALDI-TOF MS: m/z: [M-H]-: for C244H308N98O137P21 ealed.: 7455.15; found: 7456.99.

Integrin activation by the nucleic acid origami modular device

[0555] Phospho-FAK (Tyr397) cellular assay: phospho-FAK assay is based on a Homogeneous Time- Resolved FRET (HTRF®) sandwich immunoassay format comprising two specific monoclonal anti-FAK antibodies, one labeled with Europium- cryptate (donor) and the other labeled with d2 (acceptor). Upon integrin pathway activation, FAK is phosphorylated on Tyr397 leading to a close proximity between the dyes. In this configuration, the excitation of the donor with a light source triggers a Luminescent Resonance Energy Transfer (LRET) towards the acceptor, which in turn fluoresces at a specific wavelength (665nm). The specific signal modulates positively in proportion to phospho-FAK (Tyr397). FAK reagents were purchased at CisBio bioassays (Marcoules, France).

Cell culture

[0556] MCF-7 cells (Michigan Cancer Foundation - 7) were grown in RPMI supplemented with 10% FBS (without antibiotics) at 37°C, 5% CO2. Cells were split twice a week in 75 cm ² flasks. It must be specified that MCF-7 cells grow in clusters and are therefore difficult to dissociate with Versene, making the calculation of the number of cells inaccurate. MCF-7 cells were starved for twenty-four hours in RPMI before performing the experiment.

[0557] The day of the assay, cells were detached with 5ml Versene 1X solution (Gibco) for 10-15 minutes at 37°C, 5% CO2. After adding 5ml RPMI, cells were centrifuged for 5 minutes at 300 x g. The cell pellet was resuspended in RPMI in order to have the required number of cells (optimal was 10,000 - 20,000 cells,) in 4pl of medium. 4pl of cells (10,000 - 40,000 cells/well) were plated in a 96-well white plate low volume (CisBio bioassays, Marcoules, France) and mixed with 4pl of experimental and control DNA constructs stored in Folding Buffer 1X (5mM Tris-HCI pH7.8, 1 mM EDTA, 18mM MgCI2). FB1X was supplemented with 100mM NaCI and 20mM MgCI2 extemporaneously. Cells were then lysed by adding 4pl of supplemented lysis buffer 4X and incubated for at least 30 minutes at room temperature under shaking. It was found that between 5 to 30 minutes after incubation of cells with cRGD-Nucleic acid origami modular devices was sufficient to observe FAK phosphorylation.

[0558] Finally, 4pl of premixed antibody solution (vol/vol) prepared in the detection buffer were added to lysed cells and incubated 2 hours at room temperature. HTRF readings were collected using a PHERAstar plate reader (BMG Labtech) at two specific wavelengths: 665 nm for the acceptor (A) and 620 nm for the donor (D). Phospho-FAK signal was assessed by calculating the ratio R(IA/ID)-R0. R0 is the background signal from the antibodies alone that was removed to the signal measured on lysed cells. The effect of nucleic acid origami modular devices was tested on MCF-7 cells in both autonomous and remote configurations, nucleic acid origami modular devices were incubated with MCF-7 cells in suspension prior to addition of 30pM of extension oligonucleotides. No significant difference in FAK phosphorylation was detected between either configuration. Controls of extension oligonucleotides alone, with cRGD- conjugated oligonucleotides, with unmodified nucleic acid origami modular devices lacking any ligands, or with nucleic acid origami modular devices decorated with RGE molecules were also performed without significant FAK phosphorylation detected.

BtuB protein purification and conjugation

[0559] Wild-type BtuB was modified with both a cysteine substitution at the third residue in the mature chain (T3C) and a 23-residue N-terminal extension inserted between residues 4 to 5 consisting of a 6-His tag and Thrombin cleavage site. This extends the N- terminus of BtuB by approximately 8nm, providing a cysteine to reversibly attach thiolated oligonucleotide, and allowing purification of the protein by affinity chromatography. BtuBT3CHis was expressed from pBAD22 vector in BL21 (DE3) Omp8 cells, to exclude contamination from outer membrane channel proteins, was grown in LB media supplemented with 10Opg/ml ampicillin at 37°C to GD600 ~0.5 then induced with 0.2% (w/v) arabinose then grown three additional hours. Cells were pelleted and resuspended in buffer A (50mM Tris-HCI, pH=7.8, 50mM NaCI, 5% glycerol), supplemented with 1 mM PMSF, lysed by sonication, cleared by centrifugation at 3,000xg, 15 minutes, 4°C, and membrane collected by centrifugation at 40,000xg, 30 minutes, 4°C. Membranes were resuspended in buffer A and solubilized at 3mg/ml for 1 hr at room temperature with 1% Triton-X-100. Membranes were pelleted, resuspended in buffer A, and solubilized overnight at 3mg/ml at 4°C with 1% LDAO. Membrane was pelleted by centrifugation and supernatant was loaded onto 5ml HisTrap column (GE Healthcare) equilibrated in buffer A. Protein was eluted using buffer B (50mM Tris-HCI, pH=7.8, 300mM NaCI, 5% glycerol, 600mM imidazole). Approximately 48pM BtuBT3CHis was incubated with 80pM thiol-oligo for 15 minutes at room temperature with 0.6mM TCEP. Samples were then incubated with 1.4 mM copper phenanthroline for 15 minutes, then immediately injected onto a Superdex 200 HR 10/30 column equilibrated in buffer A supplemented with 0.1% LDAO. Fractions were collected and evaluated on 4-20% SDS-PAGE with and without 1 mM TCEP.

Planar Lipid Bilayer [0560] Planar bilayers composed of 60mg/ml azolectin in decane solution were painted across a 0.2-mm aperture in a two compartment chamber containing a symmetrical solution of 1 M KCI, 12mM MgCI ₂ , 1 mM EDTA, 5mM CaCI2, 20mM HEPES, pH = 7.3 except with 22mM glycerol in the cis compartment and maintained at a constant voltage. Ag-AgCI electrodes were inserted into solutions containing 1 M KCI and were connected to the measurement chamber via agar salt bridges. Data was recorded on Digidata 1440A with Axoscope and analyzed with ClampFit (version 10.2 Molecular Devices). BtuBT3CHis-oligo was added to the cis chamber at about 15nM. Addition of nucleic acid origami modular devices (nano-winchs) (97nt connectors) with complementary anchor strands were added at about 10 nM and allowed to incubate for 20 minutes before addition of extension oligonucleotides at about 400nM final concentration. DTT was added to 5mM to detach the nucleic acid origami modular device from BtuB.

Quantification of nucleic acid origami modular device bound to MCF-7 cell

[0561] To determine the binding level of nucleic acid origami modular device (nanowinch) per cell, the MCF-7 cells were incubated with a fluorescently labeled nucleic acid origami modular device to provide a quantitative fluorescence intensity distribution for the cells. This was achieved by incorporating an Alexa Fluor 488-labeled DNA staple strand directly into the backstop of the nucleic acid origami modular device, (5’-end Alexa Fluor- 488, IDT Integrated DNA Technologies ALEXA-488 AGACAAAAGGGCGACAGGTTTACCAGCGCC-3 ’(SEQ ID NO: 409)). 100 pL MCF-7 cells at 1 x 105 cells/mL suspended in RPMI were mixed for 10 minutes at 37°C with fluorescently labeled nucleic acid origami modular device at saturating concentrations in triplicate, with controls (cells only, fluorescently labeled nucleic acid origami modular device without cRGD and nucleic acid origami modular device without Alexa Fluor 488-labeled DNA staple strand). Subsequently, cells were washed twice with 10 mM PBS before it was measured using a flow cytometer. The fluorescence intensity of cells was determined using a BD Biosciences flow cytometer equipped with a 488 nm argon laser and the mean fluorescence intensity (MFI) was calculated, (BD Biosciences version 1.0 software). A sample of unlabelled nucleic acid origami modular device was used to measure the baseline auto fluorescence in the flow cytometer detectors. To determine the numbers of nucleic acid origami modular device present per cell, we used a commercially available quantification assay beads QIFIKIT (Agilent Technologies, Germany) which contains five bead populations coated with increasing but defined numbers of surface Alexa-488 fluorophore. QIFIKIT calibration and setup beads were performed according to the manufacturer’s instructions; 100 pL bead suspension was added to 3 mL PBS 0.1% (w/v)-BSA and the resulting mean fluorescence intensity of each population was analyzed. The bead populations (log) MFI was correlated with the (log) number of fluorophores per bead and used to calculate the parameters of linear regression and provide the equation: (log) fluorophore per bead = (slope of line) x (log) sample MFI + (log) fluorophore per bead value when the mean fluorescence intensity is zero. The (log) number of nucleic acid origami modular device per cell was then calculated from the (log) MFI of the sample using the regression equation.

Flow cytometry.

[0562] To determine the binding level of nucleic acid origami modular device per cell, the MCF-7 cells were incubated with a fluorescently labeled nucleic acid origami modular device to perform flow cytometry analysis and to provide a quantitative fluorescence intensity distribution for the cells. This was achieved by incorporating an Alexa Fluor 488-labeled DNA staple strand directly into the backstop of the nucleic acid origami modular device, (5’-end Alexa Fluor-488, IDT Integrated DNA Technologies ALEXA-488- AGACAAAAGGGCGACAGGTTTACCAGCGCC-3’ (SEQ ID NO: 409). It was suspended 100 pL of MCF-7 cells at 1 x 105 cells/mL in RPMI and then mixed for 10 minutes at 37°C with fluorescently labeled nucleic acid origami modular device at saturating concentrations in triplicate, with controls (cells only, fluorescently labeled nucleic acid origami modular device without cRGD and nucleic acid origami modular device without Alexa Fluor 488- labeled DNA staple strand). Subsequently, cells were washed twice with 10 mM PBS before it was measured using a flow cytometer. The fluorescence intensity of cells was determined using flow cytometry (BD Biosciences flow cytometer equipped with a 488 nm argon laser and BD Biosciences version 1.0 software), and the mean fluorescence intensity (MFI) was calculated. A sample of unlabelled nucleic acid origami modular devices was used to measure the baseline auto fluorescence in the flow cytometer detectors. To determine the numbers of nucleic acid origami modular device present per cell, we used a commercially available quantification assay beads QIFIKIT (Agilent Technologies, Germany) which contains five bead populations coated with increasing but defined numbers of surface Alexa-488 fluorophore. QIFIKIT calibration and setup beads were performed according to the manufacturer’s instructions; 100 pL bead suspension was added to 3 mL PBS 0.1% (w/v)-BSA and the resulting mean fluorescence intensity of each population was analyzed. The bead populations (log) MFI was correlated with the (log) number of fluorophores per bead and used to calculate the parameters of linear regression and provide the equation: (log) fluorophore per bead = (slope of line) x (log) sample MFI + (log) fluorophore per bead value when the mean fluorescence intensity is zero. The (log) number of nucleic acid origami modular device per cell was then calculated from the (log) MFI of the sample using the regression equation.

EXAMPLE 2: Design and assembly of the nucleic acid origami modular device (Nano-winch)

[0563] The nucleic acid origami modular device comprises a first and a second origami module in a 1 :2 trimer which are a DNA origami piston comprising an extender member and a sleeve, and two DNA origami supports (Fig. 1). The DNA origami piston consists of two domains: a 60nm long six-helix bundle (6-HB) piston topped with a 20nm stop head, and a 25nm cylinder (sleeve) folded concentrically around the piston with a about 2nm gap between them (Fig. 14), minimizing friction and allowing the two domains to slide freely. Six single-stranded scaffold connectors, up to 97 nucleic acid (nt) long, link the stop head to the top of the cylinder. Six additional 97nt connectors connect the bottom of the cylinder to the piston tip (Fig. 1 ). These single-stranded DNA connectors loops act as entropic springs with constant stiffness kDNA and exert defined stresses in the low pN range, which is mechanically translated through the nucleic acid origami modular device to the tip of the piston coupled to a molecular target with a spring of constant stiffness kprotein, a simplified model of proteins as linear springs as opposed to real proteins. The length of these connectors can be adjusted by substituting a small number of staple strands, storing the excess scaffold in reservoir loops on the backstop so the machine can be tuned cost- efficiently with defined distances and forces increasing the range of potential application. The tip of the piston positions up to three ligand moieties targeting specific cell surface receptors with precision to control both the distance between ligands (4 nm) and ligand stoichiometry allowing targeting of a single mechanoreceptor or several simultaneously at controlled distance across the about 6nm wide 6-HB piston. Two DNA origami supports, which attach to opposite sides of the cylinder through mirrored single-stranded anchors, bring the device to rest on a membrane surface, anchor it in place, and prevent toppling. The DNA origami supports provide much of the rigidity of the modular device, with a micron scale persistence length of 3.8 pm, preventing torsion on the membrane surface and hindering bending of the modular device during operation. To prevent toppling, two about 30nm 6-HBs bases project at 90° from each of the extended member of each support and 45° away from each other to lay parallel to a surface and maximize area coverage to retain an upright position. These projected 6-HB bases are each reinforced with a dsDNA transversal connector to prevent compression or bending in the supports (Fig. 1 top view). Each 6-HB base has eight anchor strand that can be functionalized with specific ligand- conjugated oligonucleotides, such as cholesterol, to localize the nucleic acid origami modular device to the bilayer and retain orientation of the device (Fig. 3).

[0564] Individual DNA origami modular device components were folded, purified by electrophoretic migration, and visualized by transmission electron microscopy (TEM) (Figs. 15 and 16). Averaged TEM particles of the DNA origami piston show the concentric folding of the cylinder around the extended member of the piston. Likewise, averages of the DNA origami support show the front, back, and side of the origami with desired folding of the 6- HB bases with the reinforcing transversal connectors. Incubating the pistomsupports with a molar ratio of (1 :2.2) and an increasing concentration of magnesium from 11 mM to 35mM enabled the assembly of the two parts to obtain a DNA origami modular device. Both the DNA origami support and piston individually migrates to a similar position on agarose gels, but samples comprising the DNA origami piston linked to one or two DNA origami support yielded two additional slower migrating bands (Fig. 2A). The upper band was purified and confirmed by TEM to be the trimeric ensemble of the DNA origami modular device (Fig. 2A, Fig. 18). Side views of the nucleic acid origami modular device confirm the proper annealing of both DNA origami supports to opposing sides of the cylinder domain of the piston. Top views of the nucleic acid origami modular device show the expected arrangement of the bases of the DNA origami supports at 90°, ideal for adhering to a membrane surface (Fig. 2A, Fig. 18).

[0565] To test the ability of the device to be addressed to a lipid membrane, it has been modified each DNA origami support with eight cholesterol moieties, for a total of 32- modifications (Fig. 19). After incubation with small unilamellar vesicles (SUVs), TEM micrographs reveal the adhesion of the modular device in the desired orientation. Interaction with smaller SUVs confirm that the modular device specifically binds to vesicles on the functionalized bases of the DNA origami supports (Fig. 3).

EXAMPLE 3: Characterization of the nucleic acid origami modular device and its autonomous activation.

[0566] It has been tested the capability of the modular device to exert mechanical stresses on a surface of interest, in particular a membrane protein. The approach has been to take advantage of the single-stranded DNA connectors behavior which functions as an entropic spring (Smith et al. Science, 271 , 795-799 (1996) - Liedl et al. Nature nanotechnology, 5, 520-524 (2010) - Mathur et al. Scientific reports, 6, 27413 (2016)) and can drive the extended member of the DNA origami piston away from the surface of the membrane. This movement is automatous and not controlled by outside signals.

[0567] First, it has been assembled the modular device with six 97nt long strand connectors between the stop head of the extended member of the DNA origami piston and the cylinder (sleeve). Measurements of the distance r between the stop head and the cylinder correlates directly to the distance d retracted on a target membrane protein as depicted in Fig. 4B. Measurements of the distance ron TEM give a distribution of distance d between about 5nm-30nm with a mean of 17.7nm (Fig. 4A). This distance distribution reveals the ability of the DNA origami piston to scan the membrane surface to recruit target proteins and then extend perpendicularly to the membrane to a maximum d of about 30nm (Fig. 4B).

[0568] To adjust the distance distribution, it has been tuned the length of the corresponding oligonucleotide reservoir loops to change this extension. Two additional designs with 60nt strand connectors (nucleic acid sequences of the staple strands to obtain 60 nt-length strand connectors consist of SEQ ID Nos: 388 to 397) and 30nt strand connectors (nucleic acid sequences of the staple strands to obtain 30 nt-length strand connectors consist of SEQ ID Nos: 398 to 407) were assembled by changing the staple strands and evaluated by TEM (Fig. 4C and 4D). The mean distribution distances of 14.0nm and 9.3nm were evaluated for the 60nt and 30nt device, respectively, from TEM singleparticle analysis. To estimate the amount of force applied by the 97nt connector device in autonomous mode, it has been first approximated the countervailing top and bottom ssDNA connectors as entropic springs using a Worm Like Chain model (WLC) with the modular device attached to a simplified target molecular spring, kprotein, with varying stiffness. For a typical molecular target with a spring of constant stiffness ranging from 0.1 to 20 pN/nm (Sotomayor, et al. Structure 13, 669—682 (2005), Lee et al. Nature, 440(7081 ):246-9. (2006)), it has been found that the force applied by the DNA origami modular device spans the range from 1.6 pN to 30 pN depending on kprotein (Fig. 5A-C). This mechanical behaviour was further explored using coarse-grained molecular dynamics and Monte Carlo simulations on oxDNA software (Ouldridge, et al. J. Chem. Phys., 134, 085101 . (201 1 ), Fig. 20 and 24) at experimental conditions with a constant 23°C to reduce thermal fluctuations on the DNA origami modular device and protein and with salt concentrations matching those of folding conditions to retain structural integrity. Simulations indicated a tension in a range similar to the WLC model. An estimate of the force exerted on the linear spring as a function of kprotein is given in Fig. 24. Force generated by the entropic spring behavior of the strand connectors in the autonomous mode is translated through the highly rigid body (Castro et al. Nanoscale 7, 5913-5921. (2015)) of the DNA origami modular device to the target molecule at the tip of the DNA origami piston.

[0569] As the DNA origami piston exerts force perpendicularly to the membrane of the cell, the DNA origami supports also distribute an equal and opposing force across a membrane that retains sufficient tension to keep the modular device moored on the surface (Fig. 4A). The force exerted by the modular device is affected by factors in the environment, including membrane deformation under the DNA origami supports (Mey et al. Journal of the American Chemical Society, 131 (20), 7031 -7039. (2009) showed that typical membrane indentation curves are linear and, with the piston of the nucleic acid origami modular device, are likely to follow about 1 .0A of vertical displacement with each pN of force exerted by the modular device.

EXAMPLE 4: Nucleic acid origami modular device (nano-winch) autonomous activation of mechanoreceptors on cells.

[0570] To explore this mechanical behavior and the functionality of the modular device on cells, it has been used the nucleic acid origami modular device to activate integrin membrane surface receptors on MCF-7 human breast cancer cells, which have been shown to express high concentrations of pi integrin and innumerous small integrin clusters (Kim et al. Experimental & Molecular Medicine 40, 261-270 (2008)). Upon ligand binding and mechanical stress, integrins shift conformation from inactive at about 1 1 nm to active, elongated up to about 19nm from cell surface (Ye et al. J. Cell Biol. 188, 157-173 (2010)). Reasonable estimates of the forces exerted on single and early activated integrins are in the range of about 1 and 15 pN (Morimatsu et al. Nano letters, 13(9), 3985-3989. (2013). - Zhang et al. Nature communications, 5(1 ), 1 -10. (2014) - Sun et al. Journal of Cell Biology, 215(4), 445-456. (2016)). These relatively low pN tensions are transmitted to the cytoskeleton to stimulate actin polymerization, and recruit various protein adhesions such as talin, paxillin, and focal adhesion kinase (FAK) (Yu et al. Proceedings of the National Academy of Sciences, 108(51 ), 20585-20590. (201 1 )) that in turn induce biochemical signals such as FAK phosphorylation (Cheng et al. Science advances, 6, eaax1909. (2020)). Recent studies have shown a direct proportional, linear relationship between force exerted on integrins and FAK phosphorylation (Zhou et al. Nature communications, 12(1 ), 1 -13. (2021 )). The nucleic acid origami modular device provides a tool for mechanical activation of membrane mechanoreceptors such as integrins, in this case on cells in suspension, in contrast to previous studies on substrate (Cheng et al. Science advances, 6, eaax1909. (2020) - Zhou et al. Nature communications, 12(1 ), 1 -13. (2021 )). We used the nucleic acid origami modular device having 97nt strand connectors which has a about 20nm dynamic range cf-distance allowing the tip of the extended member of the DNA origami piston to explore the membrane surface of the cell, to recruit the inactive integrin, and to sufficiently accommodate the height of active integrin. A synthetic cyclic-peptide (cyclic Arg-Gly-Asp, cRGD) was engineered to mimic integrin ligand (Fig. 21 ) (Aumailley et al. FEBS letters, 291 , 50-54 (1991 )) and three were clustered approximately 4nm apart on the tip of the DNA origami piston (Fig. 22) to promote cRGD-integrin engagement via statistical rebinding (Maheshwari et al. J Cell Sci, 113, 1677-1686 (2000)).

[0571] The mechano-chemical signal conversion was monitored using the cytoskeletal protein FAK that auto-phosphorylates at position Y397 upon integrin activation under force exertion (Schlaepfer et al. Progress in biophysics and molecular biology, 71 , 435-478 (1999)) (Fig. 3A). This finding is supported by other recent studies showing a direct relationship between the force applied to integrin and FAK Y397 phosphorylation (Zhou et al. Nature communications, 12(1 ), 1 -13. (2021 )), although with cells on a substrate, whereas here the cells are in suspension. We examined mechanical stimulation of integrin by measuring Luminescence Resonance Energy Transfer (LRET) between donor and acceptor fluorescently labelled anti-FAK antibodies which specifically bind FAK and phosphorylated FAK, respectively. cRGD-oligo alone and cRGD functionalized DNA origami piston without DNA origami supports showed a low LRET acceptor-donor (A-D) emission ratio response, R(A/D) - R0, found to be around 0.40x104. Fully assembled nucleic acid origami modular device lacking cRGD functionalization or functionalized with a RGE peptide as a control also showed low LRET responses, R(A/D) - R0=0.49x104 ± 0.12x104 and 0.27x104± 0.01x104, respectively. However, only fully assembled nucleic acid origami modular device functionalized with cRGD peptides and incubated with MCF-7 cells elicited a high LRET response, R(A/D) - R0=1.23 x104± 0.26 x104 (Fig. 6B). This demonstrates that only a cRGD-functionalized fully assembled nucleic acid origami modular device can interact specifically with integrin cell receptors and generate linear motion and a corresponding compressive force on the membrane through the DNA origami supports. The average number of DNA origami modular devices on cell surfaces was quantified by flow cytometry as an average of 9,200 ± 312 nucleic acid origami modular devices per cell. Under optimal MCF-7 cell densities that overexpress integrins and optimal incubation time, the numbers of DNA origami modular devices per cell generated sufficient tension to stimulate detectable downstream phosphorylation of FAK. EXAMPLE 5: Remotely controlled nucleic acid origami modular device activation

[0572] The autonomous mode of the nucleic acid origami modular device exerts an aboutl pN to 25 pN tension on the target protein depending on the spring constant kprotein, which covers a large number of biological processes (V. P. Y. Ma et al. Small, 15(26), 1900961 . (2019)). However, other molecular systems may require finer control of distance d extension and higher amounts of force to elicit a response. To achieve this, it has been explored the capabilities of the nucleic acid origami modular device to be remotely controlled by addition of oligonucleotides complementary to the strand connectors linking the stop head and the cylinder.

[0573] This movement is therefore activated by an external signal such as a “remote”. Annealing of the oligonucleotides to these strand connectors transitions them from single-stranded, with a persistence length (l _P ) of about 1 nm to double-stranded DNA (McIntosh et al. Biophys J 106, 659-666, (2014)) which has comparatively greater stiffness with a Ip of about 50nm at sodium concentrations above 10 mM (Brinkers, et al. The Journal of chemical physics. 130:06B607. (2009)). The nucleic acid origami modular device reservoir loops allow us to tune the length of the connectors with a length intermediate between these two Ip. We tested three tuned lengths of the strand connectors to ascertain their extension distances, 30-bp, 60-bp, and 97-bp. This corresponds to movement of the tip of the DNA origami piston by an anticipated distance d on a target membrane protein of 10nm, 20nm, and 33nm, respectively (Fig. 7).

[0574] The single-stranded version of each origami was folded, purified, then incubated with extension oligonucleotides complementary to the connector strands to “remotely activate” nucleic acid origami modular devices. Measurements under TEM show ratcheting of the stop head away from the cylinder with mean extension distances d of 10.7nm, 19.0nm, and 31.3nm for each device, respectively (Fig. 8A). The experimentally verified extension distances corresponded with the anticipated extension distances modelled from the WLC model (Fig. 9). Multiple double-stranded connectors in parallel increases the probability of precisely positioning the DNA origami piston to the desired extension distance. This model accurately predicts the extension distance d of the remotely activated nucleic acid origami modular device from the double stranded connector lengths and enables the user to rationally define the connectors length for a required distance d. To estimate the amount of force applied by the nucleic acid origami modular device, we performed coarse-grained molecular dynamics and Monte Carlo simulations of double stranded connectors, with respectively 60 and 97 nucleotides, using oxDNA software. For a molecular target with a spring of constant stiffness kprotein ranging from 0.1 to 20 pN/nm, it has been found the force applied by the nucleic acid origami modular device on the surface spanning the range from 1.7 pN to 80 pN (Figs. 20-24). An estimate of the force exerted on the linear spring as a function of kprotein is given in Figs. 20 and 24. For weak deformations from the equilibrium configuration, the force exerted by the nucleic acid origami modular device is proportional to the effective stiffness. The simulations encompassed by the disclosure show an effective stiffness of 8.0x10-3 N/m for double stranded and 3.0x10-3 N/m for single-stranded 97-nucleotide connectors. Note that this value could be an overestimation, as several degrees of freedom, such as relative tilting or rotation of cylinder versus stop head, were ignored.

[0575] To experimentally validate that DNA origami modular device with doublestand connectors generates tens pN-scale forces, we tested the nucleic acid origami modular devices' ability to mechanically rupture a DNA hairpin.

[0576] A DNA origami modular device with a piston-cylinder with 97-nt long strand connectors has been folded with a DNA hairpin requiring a known benchmark of force F1/2, which is defined as the equilibrium force of about 20pN (Woodside, et al. Proceedings of the National Academy of Sciences, 103, 6190-6195 (2006)) at which the hairpin spends half of its time in an unzipped state as illustrated in Fig. 10. Measurements of these tethered DNA origami piston before and after incubation with 97-nt extension oligonucleotides on TEM show extension of the modular device (Fig. 11 ). The deformation from equilibrium imposed by the presence of the hairpin can be estimated to be 5.3nm (Fig. 8A and 11). In the linear approximation, this corresponds to a 8.0x10-3 N/m x 5.3nm = 42pN force, which is larger than the hairpin F1/2. This shows that the nucleic acid origami modular device hybridization generates sufficient linear mechanical force to unzip this DNA hairpin.

[0577] It has been tested the remote activation of nucleic acid origami modular devices to mechanically open a channel. TonB-dependent receptors are p-barrel proteins with a globular plug domain occluding passage through the central channel. These proteins recruit the inner membrane protein complex TonB-ExbB-ExbD to mechanically unfold a force-labile region of the plug by approximately 20nm to form a ~13-A channel. A TonB- dependent receptor, BtuB, was engineered with an accessible 8nm linker on the plug domain containing a cysteine, allowing covalent conjugation of a thiolated-oligonucleotide complementary to an anchor strand positioned on the tip of the piston (Fig. 12) and permits detachment of the nucleic acid origami modular device upon addition of the reducing agent. The plug domain of BtuB refolds after channel opening and will presumably refold after detachment from the nucleic acid origami modular device. After BtuB purification, coupling between BtuB and thiolated-oligo leads to about 80% yield (Fig. 23). BtuB-oligo was separated from excess oligo using size exclusion chromatography and then reconstituted into planar lipid bilayers to detect channel formation via conductance across the membrane.

[0578] The presence of 4M urea elicited channel activity from the BtuB-oligo complex, confirming that plug denaturation and integrity of the engineered BtuB is similar to wild-type protein (Udho, et al. Proceedings of the National Academy of Sciences, 106, 21990-21995 (2009)). To accommodate both the 20nm extension distance (Hickman, et al. Nat. Common, 8, 14804 (2017)) and the about 8nm N- terminal linker, it has been employed the nucleic acid origami modular device with a 33nm extension from the 97-nt connectors, nucleic acid origami modular devices bearing complementary anchors to the BtuB-oligo were added to the cis-compartment with no modification of the conductivity at 4 ± 4pA. After 20 minutes complementary oligonucleotides were added in the cis compartment. Channel opening events shifted the conductivity from the closed state to an open state of 16 ± 4pA, likely reflecting the opening of the BtuB channel by the nucleic acid origami modular device (Fig. 13A). Addition of dithiothreitol to the cis chamber resulted in closing events reverting from an open state of about 20 ± 6pA to the closed state of about 8 ± 6pA (Fig. 13B). This is interpreted as the reduction of the disulfide bond between BtuB and the thiolated oligo, detaching the nucleic acid origami modular device and relaxing the plug domain to close the p- barrel. Control experiments in the absence of nucleic acid origami modular device or without the addition of extension oligos failed to elicit increased conductivity.

EXAMPLE 6: Discussion

[0579] The nucleic acid origami modular device is a tunable, adjustable nanodevice able to interface directly with molecular systems and cells to elicit biochemical effects via mechanical tension. This provides a method that expands the existing techniques to investigate mechanical-chemical communication of cells. The modules of the device are easy to assemble into a complete modular device, robust enough to be used in a variety of systems and able to adjust the applied distance and tension. Functionalization of the DNA origami supports with cholesterol is a general method to attach to a membrane bilayer. However, it is possible to functionalize with other ligands, such as other lipids, antibodies or nanobodies, to target specific cell types. Furthermore, functionalization of the DNA origami piston with controlled stoichiometry and vicinity will provide opportunities for interrogating different membrane proteins and multivalent interactions. The DNA origami method produces billions of individuals, functionalized nano-devices able to work in parallel to mechanically activate membrane proteins.

[0580] Further, the nucleic acid origami modular device may be combined with existing techniques, such as super-resolution microscopy, to explore the mechanical force landscape of living cells. Future improvements to the nucleic acid origami modular device could direct it to particular microdomains of the cells to help investigate specific cell mechanical processes. The nucleic acid origami modular device operates at a low pN range of force exertion suitable for a majority of biological effects (Ma et al. Small, 15(26), 1900961. (2019), Song, et al. Science. 357 (3377) (2017)). To achieve higher tension exertion required for other biological investigations, the device of the disclosure can be modified to generate actuation through cooperative oligonucleotide annealing (Blanchard, et al. Nano letters. 19 6977-86. (2019)), external magnetic (Maier et al. Nano Lett. 16 906- 910 (2016)), or electric fields and light (Kuzyk, et al. Nat. Common. 7, 10591 (2016)). Efforts to stabilize DNA origami devices in biological environments using covalent cross-linking or chemical modifications are ongoing (Gerling, et al. Science advances, 4(8), eaau1157 (2018), Gerling, et al. Angewandte Chemie, 131 (9), 2706-2710 (2019)). A nucleic acid origami modular device hardened against degradation in a biological environment could target specific cell types and receptors to investigate the effects of mechanical activation on, for example, different processes during specific stages of growth or development. We envision that in the near future the device of the disclosure could complement investigations into mechanotransduction systems in situations inaccessible to existing techniques.

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