DNA origami trapping agents for large viruses

Abstract

The present invention relates to an open shell based on three-dimensional polynucleotides for encapsulating a virus or a viral particle, to compositions comprising mixtures of such open shells based on three-dimensional polynucleotides, to compositions comprising a virus or a viral particle encapsulated by such open shells based on three-dimensional polynucleotides, and to methods for encapsulating viruses, viral particles or subviral particles by using such open shells based on three-dimensional polynucleotides.

Description

Technical Field

[0001] The present invention relates to a three-dimensional polynucleotide-based open shell for encapsulating viruses, virus particles or sub-viral particles, to a composition comprising a mixture of such three-dimensional polynucleotide-based open shells, to a composition comprising viruses, virus particles or sub-viral particles encapsulated by such three-dimensional polynucleotide-based open shells, and to a method for encapsulating viruses, virus particles or sub-viral particles by using such three-dimensional polynucleotide-based open shells. Background Art

[0002] Viral infections cause millions of deaths globally each year, causing great suffering and morbidity, and imposing a huge social and economic toll in terms of healthcare costs, lost working hours, and other less tangible burdens such as mental health problems associated with the loss of parents, children, and caregivers, or stigmatization. Climate change and global migration are expected to increase the threat of viral outbreaks as vectors spread to areas where they could not survive until now. The burden of viral infections will further increase due to human encroachment on habitats, urbanization and megacities with increasing population density, increased travel not only locally but also over long distances, and many other drivers of disease emergence. 41 Viruses are the pathogen class most likely to adapt to new environmental conditions because of their short generation times and genetic variability that allows for rapid evolution. 42 For most viral diseases (accounting for ~70% of the viruses on the current WHO list), there are no effective treatments. The few existing antiviral therapies are almost entirely virus-specific and do not allow for application against emerging pathogens. In addition, antiviral therapies typically face the challenge that treatment must be started very soon after infection to be effective before the viral load becomes too high and causes disease symptoms. The threat of emerging viruses requires a rapid response, but there are currently no widely applicable off-the-shelf antiviral drugs.

[0003] In this context, it is useful to first consider how current antiviral therapies work. Existing antiviral drugs target virus-specific proteins (mainly polymerases) or target essential viral or cellular structures that enable viruses to replicate and spread. The major targetable steps in the viral replication cycle are: (1) docking of the virus particle to the cell membrane of the host cell; (2) uptake into the host cell; (3) release of the viral capsid into the cytoplasm and transport of the viral genome to the replication site; (4) synthesis of viral nucleic acids and proteins and post-translational processing of viral proteins; (5) assembly of the viral components into new virus particles; (6) release of newly formed viruses from the infected cell. Most clinically available antiviral drugs are polymerase inhibitors specific for a given viral enzyme. Examples include acyclovir, which is active against herpes simplex and varicella-zoster viruses. 43Tenofovir, which is active against hepatitis B virus (HBV) and HIV, and sofosbuvir, which is active against hepatitis C virus (HCV). Examples of drugs targeting different stages of the viral life cycle are: enfuvirtide 44 , which inhibits HIV fusion (stage 2); amantadine 45 , which inhibits the uncoating of influenza A virus (stage 3); or the neuraminidase inhibitor oseltamivir 46 , which interferes with the release of influenza virus from host cells (stage 6) 46 . However, these drugs can only act when the virus replicates or spreads, but cannot kill or neutralize the virus. None of these antiviral drugs are widely applicable.

[0004] Viruses come in many shapes and sizes. Their size ranges from the scale of 10 nm to 1000 nm. For example, adeno-associated virus (AAV) is a rather small icosahedral non-enveloped virus, with a diameter of about 20 nm per particle and is reproducible. Influenza virus is an enveloped and medium-sized virus, with its size on the scale of 80 nm to 150 nm. Influenza virus is also polymorphic, which means that the particles can be in various shapes and sizes, including spherical, peanut-shaped or even filamentous. Mimivirus is representative of larger viruses with a diameter of ~700 nm.

[0005] For all viruses, attachment to the host cell membrane is a prerequisite for cell penetration, infection and replication.

[0006] Preventing virus entry into cells is increasingly being considered for the development of antiviral therapies. Examples of virus entry inhibitors include peptides 1 , antibodies 2 , dendrimers 3–5 , nanoparticles and polymers coated with virus-binding moieties 6,7 . Most of these entry inhibitors act molecule-to-molecule, which means that one antiviral agent targets one viral surface protein. Recently, the multivalent antiviral concept has been proposed, which displays multiple virus-binding molecules in complex geometries, aiming to match more mesoscale structural aspects of the target pathogen, such as two-dimensional 8-10 and three-dimensional DNA structures 11,12For example, multivalent virus - coated nanostructures provide additional options for taking full advantage of the avidity effects associated with multivalent interactions between antiviral agents and viruses. Multivalent binding results in an exponential increase in binding strength with valence and enables nearly irreversible target binding with individual weak and reversible virus binders. Thus, in the case of multivalent antiviral agents, surface alterations of the virus that reduce the binding strength of individual binders, such as those caused by antigenic drift, may be less problematic compared to monovalent binders. It is also conceivable that the virus - binding moieties used in the multivalent nanostructures themselves do not necessarily need to have neutralizing activity, since entry inhibition will be achieved at least in part by the virus - surface - blocking material of the DNA nanostructure.

[0007] It has previously been found that icosahedral DNA - origami half - shells 11 can phagocytose and neutralize viruses up to 85 nm in diameter by mechanically blocking binding interactions with the cell surface and thus prevent infection of host cells. Since there are many highly relevant larger human viral pathogens (such as, for example, influenza, coronaviruses, or herpesviruses), attempts have been made to expand this method to also target these pathogens. Influenza viruses are enveloped viruses on the scale of 80 nm to 200 nm in size and come in various shapes, including spherical, peanut - shaped, and filamentous. 13 However, previously developed phagocytic virus - shell prototypes are limited in size and shape and cannot accommodate such virus particles, or are too cumbersome to produce for use in the real world.

[0008] The genomes of viruses often mutate, which can lead to a reduced success rate of treatment regimens (such as vaccination) or even cancellation. Thus, there is a great need to allow for rapid adaptation to emerging developments, such as therapeutic interventions regarding the infectivity of a given virus. None of the above - mentioned methods are modular and flexible enough to enable the structure to quickly adapt to viral mutational changes.

[0009] Therefore, although different methods for treating viral infections have been developed or proposed to date, there is still a need to develop a concept for a general antiviral drug platform for targeting various viral pathogens. In particular, a concept that does not rely on prior detailed knowledge of the genetics and characteristics of the target virus is desirable. Additionally, it is particularly important to develop an antiviral drug platform suitable for large - scale production. Summary of the Invention

[0010] The object of the present invention is to provide constructs capable of encapsulating viruses, virus particles, or sub - viral particles. The prior art has not taught or proposed a solution to this problem, namely, using simple macromolecular building blocks (such as DNA - based nanostructures).

[0011] Thus, in one aspect, the present disclosure provides a three-dimensional polynucleotide-based open shell [1] (Figure 26) that encloses a cavity [2] and includes an opening [3] for accessing the cavity, comprising: an n-sided pyramid [4] formed by n identical replicas of an acute isosceles triangular prism t1 [5] of a first type, where n is an integer selected from 7, 8, 9, 10, 11, 12, 13, 14, and 15, wherein the base plane [6] of each prism points to the outside of the open shell, and the upper plane [7] points to the cavity, wherein two large side planes [8, 9] of each prism contain a first pattern

[10] and a second pattern

[11] of one or more protrusions and / or one or more receiving portions, wherein the first pattern and the second pattern are complementary to each other, and wherein the small side plane

[12] includes a third pattern

[13] of one or more protrusions and / or one or more receiving portions; wherein the acute isosceles triangular prism of the first type is a self-assembled DNA-based structural unit that contains between 7,500 and 10,500 base pairs.

[0012] In another aspect, the present invention relates to a three-dimensional polynucleotide-based open shell according to the present invention for use in treating a patient infected with, suspected of being infected with, or at risk of being infected with a virus, viral particle, or subviral particle.

[0013] In another aspect, the present invention relates to a composition comprising a mixture of three-dimensional polynucleotide-based open shells according to the present invention, wherein the mixture comprises three-dimensional polynucleotide-based open shells having an n value in the range of 7 to 15.

[0014] In another aspect, the present invention relates to a composition according to the present invention for use in treating a patient infected with, suspected of being infected with, or at risk of being infected with a virus, viral particle, or subviral particle.

[0015] In another aspect, the present invention relates to a method for encapsulating a virus, viral particle, or subviral particle, comprising the steps of: providing a three-dimensional polynucleotide-based open shell according to the present invention; and contacting the three-dimensional polynucleotide-based open shell with a medium comprising or suspected of comprising the virus, the viral particle, or the subviral particle.

[0016] In another aspect, the present invention relates to a method for treating a patient infected with a virus, viral particle or subviral particle, suspected of being infected with a virus, viral particle or subviral particle, or at risk of being infected with a virus, viral particle or subviral particle, comprising the step of administering to said patient a three-dimensional polynucleotide-based open shell according to the present invention or a composition according to the present invention.

[0017] In another aspect, the present invention relates to a method for treating a patient infected with a virus, viral particle or subviral particle or suspected of being infected with a virus, viral particle or subviral particle, comprising the step of contacting said patient or the body fluid of said patient with a three-dimensional polynucleotide-based open shell according to the present invention or a composition according to the present invention.

[0018] In another aspect, the present disclosure provides a composition comprising a virus, viral particle or subviral particle encapsulated by a three-dimensional polynucleotide-based open shell according to the present invention or by a three-dimensional polynucleotide-based open shell from a composition according to the present invention.

[0019] The present disclosure contemplates all combinations of any one or more of the above aspects and / or embodiments, as well as combinations with any one or more of the embodiments set forth in the detailed description and examples.

[0020] Other features, objects, and advantages of the compositions and methods herein will be apparent from the specification and drawings and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows a C10 pyramid DNA origami design (an n-sided pyramid as defined in claim 1.a, where n = 10). (A) Left: Schematic model of a C10 vertebral shell assembly. Cylinders represent single DNA double helices. Each pyramid is designed to contain ten isosceles triangle subunits. Right: Schematic of a C10 pyramid covering a viral particle. (B) Principle model of subunit design, as implemented with multilayer DNA origami in a square lattice packing. Arrows indicate shape-complementary docking sides located on side 1 and side 2 (S1 and S2). (C) The 3D electron density map determined by single-particle cryo-electron microscopy reveals a close agreement between the designed overall shape and the actual overall shape of the wedge-shaped subunit (see FIG. 9 for cryo-EM 3D class averages and field micrographs).

[0022] FIG. 2 shows the characterization of the pyramid assembly. (A) Laser-scanned fluorescence image of a 1% agarose gel on which the pyramid assembly reaction mixture was electrophoresed and samples were taken at the indicated time points. The concentration of the wedge-shaped subunit was 5 nM, the incubation temperature was 40 °C, and the solution contained 25 mM MgCl 2. M: Marker lane. Sc: M13 - 8064 scaffold as reference. (B) Exemplary negative - stained TEM micrograph showing a view of the cone assembly. Inset: Schematic illustration of the typical orientation where the cone is attached to the TEM support grid. Scale bar: 100 nm. (C) Two - dimensional TEM class averages of different cone assemblies (1) with base attachment orientations. Scale bar: 50 nm. (D) Inner diameter measurements of the 2D class averages for each cone and their frequencies of occurrence. (E) Cryo - EM view of the view micrograph showing different orientations of the cone. Scale bar: 100 nM. (F) Cryo - EM 3D reconstruction of C9 and C10 cones, with inner diameter and depth measurements.

[0023] Figure 3 shows stable cone assemblies for future in - vivo applications. (A) Schematic illustration of the stabilization workflow: UV spot - welding, oligo - lysine - PEG coating, and glutaraldehyde cross - linking coating. (B) Design schematic showing details of the strand diagram of the wedge subunits to indicate the positions of additional thymines (yellow dots) for UV spot - welding of the t1 subunit. Using caDNAno v0.2.4. 38 Blue: backbone, gray: staple strand. (C) Laser - scanned fluorescence image of a 1% agarose gel on which the cone assembly reaction mixture was electrophoresed, which had been exposed to irradiation with 310 nm light for the indicated times. The gel was run in 3 mM MgCl 2 , which is the condition where non - cross - linked cones immediately disassemble into wedge subunits (e.g., see the control or 0 - minute lane). Inset: Enlargement of the high - molecular - weight cone assembly product, with each band attributed to a closed cone with the indicated wedge subunits. (D) Exemplary negative - stained TEM images of unirradiated (and thus unstabilized) cones intercepted relative to irradiated cones under conditions of the indicated MgCl 2 concentration. Scale bar: 100 nm. (E) Exemplary negative - stained TEM images of UV spot - welded cone assemblies intercepted with only DNase I (0.001 U / μL) treatment compared to samples additionally coated with oligo - lysine - PEG (1:0.6, p:n ratio) and chemically cross - linked with glutaraldehyde. Scale bar: 100 nm.

[0024] In this context, it should be noted that with respect to FIGS. 3B, 24, and 25 (see below), these figures show schematic representations of a portion of the complex arrangement of different oligonucleotides that form the polynucleotide-based open shells of the present invention. All of the oligonucleotides used in forming these polynucleotide-based open shells are listed in Tables 1 to 3 and are included in the sequence listing. Thus, Tables 1 to 3 include all of the sequence information required to generate the nanostructures schematically shown in FIGS. 3B, 24, and 25, which are for illustrative purposes only. No additional sequence information is included in these figures.

[0025] FIG. 4 shows the phagocytosis of influenza virus particles having cones. (A) Schematic illustration of how to functionalize the cone with a viral binding moiety. Red: single-stranded DNA extension called the "stalk". Blue: antibody to the DNA tag. (B) Influenza A / PR / 8 / 34 virus capture agent having a vertebral assembly characterized by six CR9114 antibody replicates per wedge subunit. Negative staining TEM images of individual virus particles covered with different numbers of cones. Depending on the size and overall shape of the virus particle, up to three cones fit to cover the whole of the spherical / peanut-shaped virus, and even more cone replicates are adapted to cover filamentous influenza particles. Scale bar: 50 nm. (C) Negative staining TEM image of cone fitting to capture more than one virus particle at a time. Scale bar: 50 nm. (D) Section of a single-particle 3D tomogram of an influenza virus completely phagocytosed by two cones in a sandwich-like assembly obtained by negative staining TEM tilt series. Scale bar: 25 nm.

[0026] FIG. 5 shows spike cone assemblies with enhanced surface coverage. (A, B) Schematic model of the spike cone design using a second wedge block (t2), which is designed to assemble onto the base of the cone. (C) Exemplary negative staining TEM micrographs of the spike cone assembly in different clear views. (D) Exemplary TEM micrographs showing influenza A / PR / 8 / 34 virus particles phagocytosed in spike cone assemblies functionalized with 6-fold CR9114 antibody per wedge subunit. (E) Negative staining 3D TEM tomogram of an individual influenza virus particle completely phagocytosed by a single spike cone (obtaining better surface coverage than non-spike cones). All scale bars: 50 nm.

[0027] FIG. 6 shows a schematic representation of the design parameters for t1 and t2. (A) Cross-section of a 3x6 DNA helix in a square lattice array, in a straight configuration and a tilted configuration. (B) Representation of the corner angles (α and β) and lengths (a x and b x ) of the reference helix. (C) Representation of the single-stranded DNA loop bridging corner design. (D) Representation of the bevel angle corner design.

[0028] Figure 7 shows the cryo-EM determination of version 1 of t1. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) 3D histogram representing the particle orientation distribution. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

[0029] Figure 8 shows the cryo-EM determination of version 2 of t1. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) 3D histogram representing the particle orientation distribution. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

[0030] Figure 9 shows the cryo-EM determination of version 3 of t1. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) 3D histogram representing the particle orientation distribution. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

[0031] Figure 10 shows the cryo-EM electron density map of the t1 and t2 triangles. Cryo-EM was used to verify the DNA origami design during the iterative process. It allowed the twist of the first version to be corrected to an almost twist-free object (final version).

[0032] Figure 11 shows the negative stain TEM of the t1 folding reaction crude product. The micrograph shows how the t1 triangles start to assemble into cones during the folding reaction. Additional staples from the folding can be seen in the background. Scale bar: 100 nm.

[0033] Figure 12 shows the negative stain TEM of the non-specific stacking of cones induced by high ionic strength. Side and top views of the non-specific cone stacking. Scale bar: 100 nm.

[0034] Figure 13 shows the 2D class averages of the cones extracted from the negative stain TEM. The vertex-attached cones have a larger diameter and a worn perimeter compared to the base-attached cones containing the same number of wedge building units. Scale bar: 100 nm.

[0035] Figure 14 shows the cryo-EM of the cones. (A) Different views of the electron density map of the C9 cone. Scale bar: 50 nm. (B) Different views of the electron density map of the C10 cone. Scale bar: 50 nm. (C) 3D histogram representing the orientation distribution of the C9 cone. (D) As in C, but for the C10 cone.

[0036] Figure 15 shows the 3D measurements of the dimensions of the cryo-EM reconstructions. (A) C9 cone. (B) C10 cone.

[0037] Figure 16 shows the multi-body analysis of object C9. (A) Nine masks (colored, semi-transparent) enclosing the reconstruction of object C9 for multi-body refinement. (B) Principal component analysis of the refined directions of individual rigid bodies from 9-body multi-body refinement. (C) Particle weight distribution along the first principal component (PC). (D) Reconstruction of two subsets of the particle ensemble. Subset 1 (orange) contains particles with weight values from -999 to 0 along PC1, and subset 2 (blue) contains particles with values from 0 to 999.

[0038] Figure 17 shows negative-staining TEM of a negative control for influenza A / PR / 8 / 34 captured by the vertebral body. The field of view shows no binding of influenza virus particles in the absence of antibody coating. Scale bar: 100 nm.

[0039] Figure 18 shows the cryo-EM determination of t2 version 1. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) Histogram representing the particle orientation distribution. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

[0040] Figure 19 shows the cryo-EM determination of t2 version 2. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) 3D histogram representing the particle orientation distribution. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

[0041] Figure 20 shows the cryo-EM determination of t2 version 3. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) 3D histogram representing the particle orientation distribution. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

[0042] Figure 21 shows a cylindrical representation of the features of triangle 1 and 2 assemblies. (A) The third side of t1 can be functionalized with a protrusion orthogonal to the first and second sides used for t2 assembly, which has a complementary feature in the form of a recess. (B) Representation of the dimer in two different views.

[0043] Figure 22 shows the t1-t2 dimer assembly characterization. (A) Exemplary laser-scanned fluorescence image of a 1.5% agarose gel shows that t1 assembles with t2 in a 1:1 ratio over a 2-day period, where the triangle monomer concentration is 5 nM, in the presence of 25 mM MgCl 2Incubate at 40 °C under the conditions of. Sc: M13 - 8064 scaffold as a reference. The first and second sides of t1 are passivated to avoid cone assemblies. (B) Percentage (%) of fully assembled dimers at different time points and different MgCl 2 concentrations. % was extracted from the agarose gel, one as shown in (A). Error bars show the standard deviation of triplicates.

[0044] Figure 23 shows the broadband virus captured by the heparan sulfate - modified spike cones. (A) Schematic diagram of how to functionalize the cones with the virus - binding part. Red: single - stranded DNA extension called the "stalk". Orange: HS polymer. Capturing with spike cones is characterized by 12 heparan sulfate moieties per wedge - shaped subunit. (B) Exemplary negative - stain TEM micrograph showing captured SARS - COV - 2 and Zika virus - like particles (VLPs). (C) Negative - stain TEM micrograph showing captured Chikungunya VLPs. Due to the small size of CHIK - VLPs, up to three virus particles are packed into the large cavity of the spike cone, and they are severely deformed to maximize contact with the virus. All scale bars: 50 nm.

[0045] Figure 24 shows the caDNAno design diagrams for triangle 1 (A) version 1, (B) version 2, and (C) version 3. Blue: backbone, colorful: staple strands. Designs prepared using caDNAno version 0.2.4.

[0046] Figure 25 shows the caDNAno design diagrams for triangle 2 (A) version 1, (B) version 2, and (C) version 3. Blue: backbone, colorful: staple strands. Designs prepared using caDNAno version 0.2.4.

[0047] Figure 26 shows a schematic representation of the three - dimensional polynucleotide - based open - shell of the present invention including the reference numbers used in the claims. Detailed Description

[0048] The present disclosure provides constructs capable of encapsulating viruses, virus particles, or sub - viral particles.

[0049] Unless otherwise defined, 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 invention belongs.

[0050] Unless otherwise noted, the terms "comprising" and "including" are used in their open and non - limiting sense herein. With respect to the latter embodiment, the term "comprising" includes the narrower term "consisting of".

[0051] Unless otherwise indicated herein or clearly contradicted by the context, in the context of describing the present invention (especially in the context of the following claims), the terms "a / an" and "the" and similar referents shall be construed to cover both the singular and the plural. For example, the term "cell" includes multiple cells, including mixtures thereof. In cases where the plural form is used for compounds, salts, and the like, this is also considered to refer to a single compound, salt, or the like.

[0052] Thus, in one aspect, the present disclosure provides a three-dimensional polynucleotide-based open shell [1] (referenced by the reference numeral in FIG. 26), which encloses a cavity [2] and includes an n-sided pyramid [4] formed by n identical replicas of a first type of acute isosceles triangular prism t1 [5], where n is an integer selected from 7, 8, 9, 10, 11, 12, 13, 14, and 15, where the base plane [6] of each prism points to the outside of the open shell, and the upper plane [7] points to the cavity, where two large side planes [8, 9] of each prism contain a first pattern

[10] and a second pattern

[11] of one or more protrusions and / or one or more receiving portions, where the first pattern and the second pattern are complementary to each other, and where the small side plane

[12] includes a third pattern

[13] of one or more protrusions and / or one or more receiving portions; where the first type of acute isosceles triangular prism is a DNA-based self-assembling structural unit.

[0053] In a specific embodiment, the DNA-based self-assembling structural unit includes between 7,500 and 10,500 base pairs.

[0054] In a specific embodiment, the molecular weight of each DNA-based self-assembling structural unit is between 4.5 MDa and 7 MDa.

[0055] In a specific embodiment, the present disclosure provides a DNA-based three-dimensional polynucleotide-based open shell.

[0056] In the context of the present disclosure, the term "polynucleotide-based open shell (DNA-based)" refers to a DNA-based nanostructure formed by a set of DNA-based macromolecules. Nanostructures similar to the DNA-based nanostructures used according to the present invention are described in detail in WO 2021 / 165528 and in Sigl et al., in the above citation.

[0057] In the context of the present disclosure, the term "DNA" refers to deoxyribonucleic acid consisting of a single strand of monomeric units called nucleotides, where each nucleotide consists of a nitrogenous nucleobase, a 2-deoxyribose moiety, and a phosphate group, and where individual nucleotides in the single strand are linked by phosphate groups that connect the OH group in the 5'-position of the 2-deoxyribose moiety to the OH group in the 3'-position of an adjacent 2-deoxyribose moiety. In specific embodiments, the nitrogenous nucleobases are independently selected from cytosine [C], guanine [G], adenine [A], and thymine [T]. In specific embodiments, one or more of the nucleobases are non-canonical bases, in particular non-canonical bases selected from the list below: modified adenosines, in particular N6-carbamoyl-methyladenine or N6-methyladenine; modified guanines, in particular 7-deazaguanine or 7-methylguanine; modified cytosines, N4-methylcytosine, 5-carboxycytosine, 5-formylcytosine, 5-glycosylhydroxymethylcytosine, 5-hydroxycytosine, or 5-methylcytosine; modified thymines, in particular α-glutamylthymine or α-putrescinylthymine; uracil or its modifications, in particular uracil, base J, 5-dihydroxypentauracil; or 5-hydroxymethyldeoxyuracil; deoxyguanosine, and 2,6-diaminopurine. Single-stranded DNA segments can interact with complementary DNA segments through the interaction of complementary nucleobases, where cytosine and guanine, and adenine and thymine are complementary to each other by forming two (A / T) and three (G / C) hydrogen bonds between the nucleobases, respectively. In the case of genomic DNA, single-stranded DNA segments can be completely complementary to each other, or may be partially complementary to each other, including cases where one single-stranded DNA is partially complementary to two or more other single-stranded DNA strands. The interaction of two complementary single-stranded DNA sequences results in the formation of a double-stranded DNA helix.

[0058] As is well known, DNA has evolved in nature as a carrier of genetic information encoding proteins. DNA further includes non-coding regions, which include regions with regulatory functions. Thus, any DNA-based application typically relies heavily on specific DNA sequences and is almost always achieved only by naming specific DNA sequences. In contrast, in the context of the present invention, such coding and / or regulatory functions play no role and may or may not be present, as the basic DNA sequences are designed and selected only in the required arrangement to form double helix subunits. Thus, in one embodiment, any form of long single-stranded DNA sequence can be selected as a template, whether naturally occurring DNA (such as DNA of a bacteriophage) or synthetically produced DNA, and a set of short single-stranded DNA sequences can be designed, where each sequence is complementary to one or more different parts of the template, thereby forming one or more double helix segments. Overall, all these double helix segments are generated by the interaction of the entire set of short single-stranded DNA sequences with the template and then form the required three-dimensional arrangement. Starting from a given single-stranded template sequence, known techniques can be used to establish the design of a set of complementary templates, such as methods for synthesizing megadalton-scale discrete objects with well-defined 3D shapes 15,40,49-60 . Specifically, an iterative design paired with elastic network-guided molecular dynamics simulations 61 of caDNAno 38 can be used.

[0059] In addition to the complementary nucleobases of different segments of single-stranded DNA interacting via hydrogen bonds, other interactions may occur between different DNA strands, including interactions between the ends of two double-stranded DNA helices by using the protruding and recessed features of blunt or sticky ends for enhancing stability and specificity 62 , enabling the design and formation of complex DNA-based nanostructures via shape complementarity of double helix subunits. Thus, the two three-dimensional arrangements formed according to the previous paragraph can interact with each other through the interaction between the double helix subunits present on the two three-dimensional arrangements, including specific interactions between two three-dimensional arrangements with complementary protrusions and recesses (or pestles and mortars).

[0060] In a specific embodiment, the DNA-based nanostructure is formed by self-assembling DNA-based structural units.

[0061] In a specific embodiment, each of the self-assembling DNA-based structural units is formed by a single-stranded DNA template strand and a set of oligonucleotides complementary to the single-stranded DNA template, where each of the oligonucleotides is complementary to a continuous DNA sequence segment on the single-stranded DNA template or to at least two non-continuous DNA sequence segments on the single-stranded DNA template.

[0062] In a specific embodiment, the DNA-based nanostructure is composed of between 4 and 180 such self-assembled DNA-based structural units.

[0063] In a specific embodiment, the single-stranded DNA template is the single-stranded DNA of a filamentous phage or single-stranded DNA derived from a filamentous phage.

[0064] In the context of the present invention, the term "filamentous phage" refers to a type of phage or bacteriophage characterized by its filamentous shape with a genome that typically contains circular single-stranded DNA and infects Gram-negative bacteria. Filamentous phages include Ff phages such as M13, f1, and fd1 phages, as well as Pf1 phage.

[0065] In a specific embodiment, the single-stranded DNA template has the sequence according to SEQ ID NO:1 (M13 8064) (see Table 1). In other specific embodiments, the single-stranded DNA template has the sequence of M13 7249 (see SEQ ID NO:2 of WO 2021 / 165528).

[0066] In a specific embodiment, the single-stranded DNA is circular.

[0067] In the context of the present invention, a single-stranded DNA template "derived from filamentous phage single-stranded DNA" refers to a DNA construct derived from a published DNA sequence of a naturally occurring filamentous phage by one or more of the following: (i) opening the circular structure into a linear sequence; (ii) deletion of one or more nucleotides; (iii) insertion of one or more nucleotides; (iii) substitution of one or more nucleotides; (iv) addition of one or more nucleotides; and (v) modification of one or more nucleotides. Although any such variations may have a harmful or at least quite unpredictable effect on phage biology, its infectivity, and its proliferative ability, these effects play no role in the context of the present invention because, as described above, the single-stranded DNA template is only used as a naked template and does not need to have any functional properties, and all structural aspects (such as the correct formation of the three-dimensional shape of the self-assembled DNA-based structural units) are achieved by the correct selection of the set of complementary oligonucleotides.

[0068] In the oligomer embodiment, the single-stranded DNA template has at least 80%, particularly at least 90%, more particularly at least 95% sequence identity with the naturally occurring or published sequences of filamentous phages, particularly with M13, f1 or fd1 phages, particularly with the sequences selected from SEQ ID NO:1 (M13 8064) and M13 7249 (see SEQ ID NO:2 of WO 2021 / 165528). In this context, it should be mentioned that the single-stranded DNA template is only used as a template in the present invention, so the exact sequence does not have any biological role and / or function. On the contrary, any sequence of a similar length can be used, because the establishment of the three-dimensional structure of the polynucleotide-based open shell is essentially achieved by synthesizing a set of oligonucleotides that are complementary to two or more sequence segments on the single-stranded DNA template. This set of complementary oligonucleotides can be designed manually, but it is easier to use a computer program (such as, for example, caDNAno 37 )). Therefore, the phage sequences listed above are given only as examples.

[0069] In the context of the present invention, the term "acute isosceles triangular prism" refers to a polyhedron in which all vertices are located in two parallel planes, which is a triangular prism having two acute isosceles triangular planes.

[0070] In a specific embodiment, the present invention relates to a DNA-based nanostructure, wherein each of the triangular prisms is formed by m triangular planes, where m is an integer independently selected from 4, 5, 6, 7 and 8, particularly an integer independently selected from 5, 6 and 7, more particularly where the integer is 6, and three or four edges of each of the m planes are respectively formed by n parallel DNA double helix segments, where n is an integer independently selected from 1, 2, 3, 4, 5 and 6, particularly an integer independently selected from 2, 3, 4 and 5, more particularly independently selected from 3 and 4, and each plane is connected to the plane above the plane and / or the plane outside the plane, (i) through the interaction between the DNA double helices forming the plane by stacking, and (ii) partly through the DNA segments within the single-stranded DNA template and / or the oligonucleotides forming the DNA-based structural units bridging at least two of the planes, and at least two of the three or four side trapezoids respectively include recesses and / or protrusions of a specific pattern formed by deleting or adding DNA double helix segments for specific interaction with complementary patterns on the side trapezoids of another self-assembled DNA-based structural unit.

[0071] In a specific embodiment, the average length of each of the n DNA double helix segments in the m planes of the triangular or rectangular prism is between 80 base pairs and 200 base pairs.

[0072] In a specific embodiment, the triangular prism is a truncated triangular pyramid.

[0073] In the context of the present invention, the term "truncated triangular pyramid" refers to a three-dimensional geometric shape in the form of a triangular pyramid, where the tip of the pyramid has been removed, resulting in a plane parallel to the base of the pyramid being present at the top.

[0074] In a specific embodiment, for at least a portion of the self-assembled DNA-based structural units, the length of at least one edge of each of the m planes gradually decreases from the first plane to the mth plane, thereby creating an oblique angle θ (see Figure 6) between the plane perpendicular to the first plane and the trapezoidal plane formed by the m edges. In a specific embodiment, all three trapezoidal planes exhibit an oblique angle.

[0075] In a specific embodiment, the oblique angle is between 16° and 26°, particularly between 18° and 24°, more particularly between 20° and 22°, and most particularly approximately 20.9°.

[0076] In a specific embodiment, the DNA-based nanostructure comprises at least one set of self-assembled DNA-based structural units, wherein all three or four lateral trapezoids respectively comprise recesses and / or protrusions of a specific pattern formed by deletion or addition of DNA double helix segments for specific interaction with a complementary pattern on the lateral trapezoid of another said self-assembled DNA-based structural unit.

[0077] In a specific embodiment, the three-dimensional polynucleotide-based open shell further comprises n replicas of a second type of acute isosceles triangular prism t2

[14] , wherein the first side

[15] of each prism points towards the outside of the open shell and the opposite side

[16] points towards the cavity and / or towards the opening for accessing the cavity, wherein one plane

[17] of the second type of prism structure

[14] comprises a fourth pattern

[18] complementary to the third pattern

[13] and comprising one or more protrusions and / or one or more receiving portions.

[0078] In a specific embodiment, the DNA-based nanostructure comprises two sets of self-assembled DNA-based structural units, particularly self-assembled DNA-based structural units t1 and t2.

[0079] In an alternative aspect of the present invention, the present invention relates to a macromolecule-based nanostructure, which is an RNA-based nanostructure.

[0080] In the context of the present disclosure, the term "RNA" refers to ribonucleic acid consisting of a single strand of monomeric units called nucleotides, where each nucleotide is composed of a nitrogenous nucleobase, a ribose moiety, and a phosphate group, and where individual nucleotides are linked in the single strand by phosphate groups that connect the OH group in the 5'-position of the ribose moiety to the OH group in the 3'-position of an adjacent ribose moiety. In a specific embodiment, the nitrogenous nucleobases are independently selected from cytosine [C], guanine [G], adenine [A], and uracil [U]. In a specific embodiment, one or more of the nucleobases are non-canonical bases, in particular non-canonical bases selected from the list consisting of pseudouracil, ribothymidine, and inosine. Unlike DNA, RNA is typically in a single-stranded form, but the formation of a double-stranded form can be achieved by the interaction of complementary nucleobases, where cytosine and guanine, and adenine and uracil are complementary to each other by forming two (A / U) and three (G / C) hydrogen bonds between the nucleobases, respectively. In a specific embodiment, the present disclosure provides macromolecule-based nanostructures, which are RNA-based nanostructures.

[0081] In the context of the present invention, the term "cavity" relates to the space enclosed by the DNA-based nanostructure. In a specific embodiment, the cavity is similar to a sphere, where a spherical segment has been cut, and the cutting plane is formed by self-assembled DNA-based structural units at the boundary of the DNA-based nanostructure. In a specific embodiment, the cutting plane is a great circle, such that the DNA-based nanostructure is a semi-shell.

[0082] In a specific embodiment, the upper plane [7] and / or, when present, the opposite side

[16] comprises one or more attachment sites for the attachment of one or more binding molecules that interact specifically or non-specifically with a virus, virus particle, or sub-viral particle.

[0083] In a specific embodiment, the one or more binding molecules interact specifically with the virus, virus particle, or sub-viral particle by being able to bind and inactivate the virus particle or the sub-viral particle.

[0084] In a specific embodiment, the binding molecule interacts specifically with a virus, virus particle, or sub-viral particle. In particular, the binding molecule is selected from antibodies and antigen-binding fragments thereof, which comprise at least the antigen-binding site of an antibody, in particular at least the VH domain of an antibody, or a combination of the VH and VL domains of at least an antibody, in particular an scFv fragment.

[0085] In a specific embodiment, the binding molecule non-specifically interacts with a virus, virus particle or sub-viral particle, in particular a construct comprising at least one sulfated or sulfonated polysaccharide group (in particular a construct comprising one or two sulfated or sulfonated polysaccharide groups), wherein the sulfated or sulfonated polysaccharide is independently selected from the list consisting of: heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate, dextrin 2-sulfate, aptamer, peptide, host-receptor domain and sialic acid.

[0086] In the context of the present application, the term "virus particle" refers to virus-like particles with a three-dimensional structure similar to that of a complete virus but without biological activity, and the term "sub-viral particle" refers to smaller virus-like particles with fewer or smaller subunits, which can be produced by not expressing all and / or only part of one or more major viral capsid proteins in certain viruses. These artificial virus particles or sub-viral particles retain the structure and antigenic properties of their native virus, including virus-specific molecular patterns and high densities of B-cell and T-cell epitopes, to induce strong innate, humoral and cellular immune responses in animals and humans, respectively. 68 。

[0087] Importantly, in addition to targeting specific receptors, many viruses also interact weakly with different biological substances, including sulfation of sulfated polysaccharides ( 63 ; see Table 4).

[0088] In the context of the present application, the term "sulfated or sulfonated polysaccharide group" refers to a group containing a polysaccharide that contains at least one sulfated hydroxyl group or at least one sulfonated sugar amino group.

[0089] Importantly, in addition to targeting specific receptors, many viruses also interact weakly with different biological substances, including sulfation of sulfated polysaccharides ( 63 ; see Table 4).

[0090] In a specific embodiment, the polysaccharide comprising at least one sulfated hydroxyl group or at least one sulfonated sugar amino group is selected from the list consisting of: heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate and dextrin 2-sulfate.

[0091] In a specific embodiment, the polysaccharide comprises at least one sulfated hydroxyl or at least one sulfonated sugar amino group, which consists of between 3 and 15 disaccharide units, in particular 4, 5, 6, 7, 8 monosaccharide units in 9 units, in particular 4 or 9 monosaccharide units.

[0092] In a specific embodiment, each disaccharide unit of the disaccharide unit contains two or three O- and / or N-sulfonic acid groups, in particular three O- or N-sulfonic acid groups.

[0093] In a specific embodiment, said polysaccharide comprising at least one sulfated hydroxyl or at least one sulfated sugar amino group is independently selected from heparin, heparan sulfate, and hybrid heparan sulfate.

[0094] In the context of the present invention, the terms "heparin" and "heparan sulfate" both refer to a family of linear sulfated heteropolysaccharides found on cell membranes and in the extracellular matrix as part of heparan sulfate proteoglycans (HSPGs). They are composed of repeating 1→4-linked disaccharide units, where one monosaccharide is an α-D-glucosamine residue and the other is a uronic acid (or the salt form of uronic acid). Heparin is a structurally similar polysaccharide present in mast cells and is a component of serum glycoprotein proteoglycans. Heparan sulfate and heparin can be defined as follows: First, in heparin, the uronic acid is mainly α-L-iduronic acid ester, while in heparan sulfate, the uronic acid is mainly β-D-glucuronic acid ester, the C-5 epimer of α-L-iduronic acid ester. Second, in heparan sulfate, the D-glucosamine residues are mainly N-acetylated, while in heparin, they are N-sulfated. Finally, although at least 70%-80% of heparin is composed of the disaccharide L-iduronic acid ester 2-O-sulfate α(1→4)D-glucosamine N,6-sulfate, in heparan sulfate, approximately 40%-60% of the disaccharides are composed of (1→4)D-glucuronic acid ester β(1→4)D-glucosamine, which can be N-acetylated or N-sulfated. These structural features together make heparin more sulfated and thus have more charges than heparan sulfate. However, it is clear that the names of heparin or heparan sulfate are not as clear as implied by this description, and polysaccharides isolated from some organisms seem to be hybrid constructs. In the context of the present invention, the term "hybrid heparan sulfate" is used to refer to such hybrids having a structure that is a mixture of "typical" heparin structural elements (L-iduronic acid ester; high sulfation degree) and "typical" heparan sulfate structural elements (D-glucuronic acid ester; N-acetylation and 6-O-sulfation).

[0095] Heparan sulfate proteoglycan (HSPG) 63；64 Commonly found on the surface of mammalian cells. The weak interaction of viruses with HSPG is conserved among virus families and thus seems to be generally beneficial for the virus life cycle. For example, the HSPG-virus interaction can enable the virus particles to perform an enhanced diffusion search for their specific host cell receptors on the cell surface. The interaction between heparan sulfate (HS) and viruses has been used for medical purposes, such as a condom virus isolation coating based on HS-modified dendrimers 3-5 . Other studies often involve the surface functionalization of nanoparticles and polymers with HS derivatives to produce virus-binding complexes with antiviral activity 6 ;7；65；66 Generally, a high level of multivalency is required to increase the binding strength between the HS nanoparticles and the virus. The reversibility of the binding may lead to the undesired dissociation and release of infectious virus from the viral isolation coating, or a high concentration of the therapeutic active agent needs to be maintained. 5 。

[0096] In a specific embodiment, the macromolecule-based nanostructure on average comprises between 1 and 10 binding molecules, particularly between 4 and 10 binding molecules, especially four, five, six, seven, eight, nine or ten binding molecules, attached to internal sites of the cavity formed by the macromolecule-based nanostructure.

[0097] In a specific embodiment, one or more of the self-assembled DNA-based structural units are linked to a construct comprising at least one sulfonated or sulfated polysaccharide group pointing into the interior of the cavity, particularly a construct comprising one or two sulfonated or sulfated polysaccharide groups.

[0098] In a specific embodiment, the three-dimensional polynucleotide-based open shell is a DNA-based nanostructure according to the invention, wherein at least one of the binding molecules is linked to one of the triangular prisms forming the DNA-based nanostructure in such a way that the at least one binding molecule is located inside the DNA-based nanostructure and points into the cavity formed by the DNA-based nanostructure.

[0099] In a specific embodiment, each prism comprises between 1 and 45, particularly between 1 and 32, such attachment sites, especially between 3 and 10 attachment sites. In a specific embodiment, all prisms comprise the attachment sites. In other embodiments, only the t1 prism comprises the attachment sites, or only the t2 prism comprises the attachment sites.

[0100] In a specific embodiment, the attachment site is a first single-stranded oligonucleotide.

[0101] In a specific embodiment, the binding molecule is attached to the attachment site by a second single-stranded oligonucleotide that is linked to one or more binding molecules and is complementary to or capable of site-specific interaction with the first single-stranded oligonucleotide. In a specific embodiment, each of the single-stranded oligonucleotides is linked to one binding molecule. In other embodiments, each of the single-stranded oligonucleotides is linked to two binding molecules.

[0102] In a specific embodiment, each of the acute isosceles triangular prisms of the first type and optionally the acute isosceles triangular prisms of the second type is a DNA-based nanostructure formed from self-assembling DNA-based structural units, wherein the DNA-based nanostructure is formed from a single-stranded DNA template strand and a set of oligonucleotides complementary to the single-stranded DNA template, wherein each of the oligonucleotides is complementary to a continuous DNA sequence segment on the single-stranded DNA template or to at least two non-continuous DNA sequence segments on the single-stranded DNA template.

[0103] In a specific embodiment, the apex angle of the acute isosceles triangle forming the opposing planes of the acute isosceles triangular prism is between 15° and 60°, particularly between 20° and 30°.

[0104] In a specific embodiment, n is an integer selected from 9, 10, 11, 12, and 13.

[0105] In a specific embodiment, the three-dimensional polynucleotide-based open shell further comprises chemical crosslinking between different prisms, which further comprises one or more crosslinks within one of the triangular prisms and / or between two of the triangular prisms.

[0106] In the context of the present invention, the term "crosslinking" refers to any permanent or intermittent connection within one of the triangular prisms and / or between two of the triangular prisms. Any such connection can be achieved a priori by connecting two oligonucleotides used to form the self-assembling DNA-based structural units prior to assembly, or can be achieved a posteriori by adding a connection, for example, chemically or photochemically, between different parts of the three-dimensional nanostructure. For example, a permanent connection can be created by photochemical crosslinking to appropriately position T residues in the structure with the formation of covalent cyclobutane pyrimidine dimer (CPD) bonds 19 , and an intermittent connection can be created, for example, by photochemical crosslinking at the blunt ends of two double-helical subunits between a 3-cyano vinyl carbazole (cnvK) moiety positioned at one blunt end and a thymine residue (T) positioned at the other blunt end 67 .

[0107] In a specific embodiment, the three-dimensional polynucleotide-based open shell further comprises chemical crosslinking between different triangular prisms.

[0108] In a specific embodiment, the chemical crosslinking is obtained by ultraviolet irradiation.

[0109] In a specific embodiment, the three-dimensional polynucleotide-based open shell further comprises an outer surface coating of the open shell with a polycationic molecule.

[0110] In a specific embodiment, the polycationic molecule is polylysine, in particular polylysine-PEG.

[0111] In a specific embodiment, the three-dimensional polynucleotide-based open shell further comprises crosslinking of the free amino groups of the polylysine, in particular crosslinking with an alkane dialdehyde, in particular crosslinking with glutaraldehyde.

[0112] In a specific embodiment, the opening [3] has a diameter

[19] between 100 nm and 200 nm.

[0113] In the context of the present invention, the term "diameter" refers to the diameter

[19] as shown in Figure 26.

[0114] In a specific embodiment, the three-dimensional polynucleotide-based open shell has a molecular weight between 30 Mda and 80 Mda (only t1), in particular between 40 Mda and 70 Mda, and between 60 Mda and 160 Mda (t1 plus t2), in particular between 80 MDa and 140 MDa.

[0115] In a specific embodiment, the volume of the cavity enclosed by the three-dimensional polynucleotide-based open shell (in nm 3 ) is between 80,000 and 200,000, in particular between 100,000 and 140,000.

[0116] In another aspect, the present invention relates to a composition comprising a mixture of three-dimensional polynucleotide-based open shells according to the present invention, wherein the mixture comprises three-dimensional polynucleotide-based open shells having an n value in the range of 7 to 15.

[0117] In particular in the range of 9 to 13, and maximally in the range of 9 to 11.

[0118] In another aspect, the present invention relates to a composition according to the present invention for use in treating a patient infected with, suspected of being infected with, or at risk of being infected with a virus, viral particle or subviral particle.

[0119] In another aspect, the present invention relates to a method for encapsulating a virus, viral particle or subviral particle, which comprises the steps of: providing a three-dimensional polynucleotide-based open shell according to the present invention; and contacting the three-dimensional polynucleotide-based open shell with a medium comprising or suspected of comprising the virus, the viral particle or the subviral particle.

[0120] In a specific embodiment, the method is for removing the virus, the viral particle or the subviral particle from the above-mentioned vehicle. In a specific embodiment, the method is used for encapsulating the virus, the viral particle or the subviral particle in order to transport the virus or the subviral particle.

[0121] In a specific embodiment, the method for removing the virus, the viral particle or the subviral particle involves a method for treating a patient infected with, suspected of being infected with, or at risk of being infected with the virus, the viral particle or the subviral particle, which comprises the step of administering to the patient a three-dimensional polynucleotide-based open shell according to the present invention or a composition according to the present invention.

[0122] In a specific embodiment, the method for treating a patient infected with or suspected of being infected with a virus, a viral particle or a subviral particle comprises the step of contacting the patient or the patient's body fluid with a three-dimensional polynucleotide-based open shell according to the present invention or a composition according to the present invention.

[0123] In another aspect, the present disclosure provides a composition comprising a virus, a viral particle or a subviral particle encapsulated by a three-dimensional polynucleotide-based open shell according to the present invention or by a three-dimensional polynucleotide-based open shell from a composition according to the present invention.

[0124] In a specific embodiment, the composition is formed during the process of removing the virus, the viral particle or the subviral particle from a vehicle containing the virus, the viral particle or the subviral particle. In other specific embodiments, the composition is formed during the process of incorporating the virus, the viral particle or the subviral particle as a cargo into the three-dimensional polynucleotide-based open shell.

[0125] In another aspect, the present invention provides a composition comprising a cargo different from a virus, a viral particle or a subviral particle, wherein the cargo (such as a complex macromolecule) is encapsulated by a three-dimensional polynucleotide-based open shell of the present invention. In a specific embodiment, the cargo is a cytokine. In a specific embodiment, the cytokine is interleukin-6.

[0126] In another aspect, the present invention provides a method for encapsulating a cargo different from a virus, virus particle or sub-viral particle (such as a complex macromolecule), which comprises the steps of: providing a three-dimensional polynucleotide-based open shell according to the present invention, and contacting the three-dimensional polynucleotide-based open shell with a medium containing or suspected of containing the cargo. In a specific embodiment, the cargo is a cytokine. In a specific embodiment, the cytokine is interleukin-6.

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[0175] It should be understood that certain features of the present invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, the various features of the present invention that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination. All combinations belonging to the embodiments of the present invention are specifically included in the present invention and are disclosed herein as if each combination and every combination were separately and clearly disclosed. In addition, all sub-combinations of the various embodiments and their elements are also specifically included in the present invention and are disclosed herein as if each and every such sub-combination were separately and explicitly disclosed herein.

[0176] The present invention is not limited to the scope of the specific embodiments described herein. Indeed, various modifications of the present invention will become apparent to those skilled in the art based on the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0177] To the extent permitted by their respective patent laws, all patents, applications, publications, test methods, literature, and other materials cited herein are incorporated herein by reference.

[0178] The following examples illustrate the invention described above, but are not intended to limit the scope of the invention in any way. Other test models known to those skilled in the art may also determine the beneficial effects of the claimed invention.

[0179] Examples

[0180] Introduction

[0181] Virus-encapsulated macromolecular shells or flatting elements can, in principle, prevent viruses from entering cells. Here, we describe the design and assembly of a cone-shaped DNA origami higher-order assembly that can engulf and flatten the surfaces of polymorphic virus samples larger than 100 nm. We determined the structure of the subunit and the complete cone assembly using cryo-EM; and established stabilization treatments to enable its use under in vivo conditions. We describe, using the cone as an example, the engulfment of influenza A virus particles and SARS-CoV-2, chikungunya, and Zika virus-like particles. Depending on the relative sizes of the cone and the virus particles, each individual cone may capture multiple virus particles, and multiple cones can also flatten and conform to the surfaces of aspherical virus particles. The cone assembly forms with high yield, requires little purification, and is suitable for large-scale production, which is a key requirement for future practical applications, including as an antiviral agent.

[0182] To overcome the limitations mentioned in the background section of the present invention, we describe here an efficiently assembled DNA origami-based macromolecular capsid system that can engulf pleomorphic viral pathogens with diameters greater than 100 nm, such as influenza A virus as an example. Our design concept considers the self-limited oligomerization of wedge-shaped building units into cones. The previous implementation of such an extension of planar finite-size assemblies used a minimal number of subunit types, thus reducing the complexity of the assembly process. The resulting high-yield assemblies enable the cone system to be mass-produced according to the actual application requirements as an antiviral drug in the future. 14 This extension of the previous implementation used the minimum number of subunit types, thus reducing the complexity of the assembly process. The resulting high-yield assemblies enable the cone system to be mass-produced according to the actual application requirements as an antiviral drug in the future.

[0183] Results and Discussion

[0184] Our cone assemblies are designed to be formed from multiple copies of wedge-shaped building units (t1) (see Supporting Information for design details). The wedge-shaped building units can oligomerize via two different self-complementary edges at opposite faces. The oligomerization of the wedges results in a self-closed circular assembly. Given the designed geometry of the wedges, we expect the cone to have ten faces (Figures 1A and 1B). The diameter of the base of the cone made of ten wedges is designed to measure ∼120 nm, and thus, two copies of the cone will be large enough to enclose influenza virus particles (∼80 - 200 nm) in a sandwich-like assembly (Figure 1A, right).

[0185] We implemented the wedge-shaped building units using multilayer DNA origami in a square lattice helical packing 15,16 . We assembled the objects using DNA origami methods and used single-particle cryo-electron microscopy (Cryo-EM) to refine and validate the design of our wedge subunits during an iterative process (Figures 7 to 9). The 3D electron density map we determined for a single wedge particle revealed the overall shape of the triangular building unit and the shape-complementary docking features (Figure 1C). Our initial wedge design showed significant global distortion, which we then corrected to an almost distortion-free shape (Figure 10).

[0186] We triggered the oligomerization of the wedge subunits by increasing the ionic strength of the solution after folding the wedge-shaped building units from their constituent DNA staples and backbone. Depending on the ionic conditions used, oligomerization can also occur simultaneously during the wedge assembly reaction (Figure 11). We monitored the formation of the cones in a time-dependent manner by gel electrophoretic mobility analysis (Figure 2A), where the appearance and disappearance of bands towards increasingly lower electrophoretic mobilities reflect the progressive oligomerization of the wedge subunits as a function of incubation time. Eventually, the oligomeric material accumulates in a relatively broad band of low electrophoretic mobility.

[0187] We imaged the final oligomeric products using negative stain transmission electron microscopy (TEM). Micrographs revealed predominantly circular structures with an appearance of vertebral shapes (Figure 2B) composed of 9, 10, 11, 12, and rarely 13 replicas of the wedge-shaped subunits, respectively. Regarding how many wedges each cone contains, the degree of heterogeneity observed in the cone oligomers may be related to the limited elasticity of the wedge-shaped structural units and their interaction interfaces. These properties can be tuned, if desired, similar to the planar ring assemblies described previously. 14 However, in the context of the current target applications, the distribution of cone products covering species with wedges in the range of 9 to 13 seems to be favorable for handling pleomorphic virus samples.

[0188] The distribution of cone products observed by TEM partly explains the relatively broad product band in gel electrophoresis analysis. We also found that under conditions of elevated magnesium concentration (such as the magnesium concentration used in gel electrophoresis), the cones have a tendency to stack on top of each other (Figure 12), which explains the blurring and formation of aggregates in high-magnesium gel electrophoresis, as shown in Figure 2A. As we further show below, there is no cone-to-cone stacking under low-magnesium conditions.

[0189] We observed three key preferred orientations of the cones in the TEM micrographs (Figure 2B, inset), including cones with their bases adsorbed on the surface (1), cones landing on the vertices (2), and cones attached to their sides (3). Compared to the base-attached cones containing the same number of wedge-shaped structural units, the vertex-attached cones have a larger diameter and a worn perimeter. It is speculated that in the vertex-attached orientation, the adhesion flattens the cone, causing the wedges to separate. In the base-attached orientation, the cone remains intact and is supported by its base.

[0190] We calculated two-dimensional (2D) class averages from the TEM micrographs, which revealed several classes corresponding to different views (Figure 13) and different cone species. Figure 2C exemplarily shows the class averages of base-attached cones with 9 to 13 wedge-shaped subunits, respectively. We measured the diameters of the undeformed base-attached particles (1) in each class average, and they match our expectations very well (Figure 2D). Thus, the C9 cone species has an average inner diameter of 110 nm. C10 has 126 nm and the largest C13 species has 147 nm. Starting from the 2D class averages, we also quantified the relative frequencies at which different cone species occur. The most abundant cone is C10 with 37% of the population, followed by C11 (27%), C9 (20%), C12 (15%), and C13 (1%).

[0191] We performed cryo-EM studies of cones in self-supporting ice to obtain 3D information of the assembled products. Exemplary cryo-EM fields of view (Figure 2E) show different orientations of partially and fully assembled cones. We determined 3D reconstructions for the C9 and C10 cone species, which confirmed the overall 3D conical shape (Figure 2F and Figure 14). The electron density maps of both cone species have elliptical undulating bases. For the C9 cone map, the ellipticity is more pronounced. We measured the lengths of the inner minor and major axes to be 100 nm and 122 nm for C9 and 114 nm and 131 nm for the C10 species (Figure 15). The cavity depth of the C9 cone is 42 nm, while C10 is shallower (39 nm). The perimeter of the base-attached cones from negative stain data and cryo-EM reconstructions in solution is in good agreement (Table 8). We hypothesize that electrostatic interactions between the cones used for negative stain TEM and the carbon surface of the grid result in a flattening effect, giving rise to a more circular ring shape. Surface interactions at the sample-air interface prior to plunge freezing may also cause particle deformation seen in the 3D maps. Cryo-EM data and reconstructions of subsets of the particle ensemble with multibody refinement and principal component analysis indicate a degree of flexibility of the cones (Figure 16), which is desirable for the intended applications.

[0192] At the salt concentrations present in physiological fluids, DNA origami higher-order assemblies (such as those presented in this work) will generally dissociate 17 . Wedge monomers are also prone to denaturation due to screened internal repulsive electrostatic forces. The physiological environment may also contain nucleases capable of degrading foreign DNA molecules by catalyzing the hydrolysis cleavage of phosphodiester bonds in the DNA backbone 18 . To make our cone assemblies persistent under in vivo-like conditions, we established a three-step post-assembly stabilization treatment, as shown in Figure 3A. The first step utilizes ultraviolet light to induce crosslinking of closely adjacent thymine bases within the DNA nanostructure 19 . Upon irradiation with a 310-nm wavelength, the double bonds of adjacent pyrimidines undergo a [2+2] cycloaddition reaction to produce cyclobutane pyrimidine dimers. To ultraviolet crosslink ("ultraviolet spot weld") the cone assemblies, we placed additional unpaired thymine bases at the helical interface of the wedge-wedge subunit interaction sites (yellow dots in Figure 3A, Figure 3B). We tested the efficacy of ultraviolet crosslinking of the cones as a function of exposure time to a 310-nm light source (Figure 3C). Once ultraviolet welding is correctly performed, the cones remain intact when exposed to low Mg 2+ concentrations, while unirradiated or under-irradiated control samples rapidly dissociate into wedge subunits (Figure 3C, Figure 3D). The ultraviolet-linked cones now appear as five distinct bands in a low ionic strength gel (3 mM MgCl 2 ).

[0193] To protect the cone assembly from nuclease-mediated degradation, we used the previously described oligolysine-PEG copolymer-based coating 20 , and then cross-linked the coating with glutaraldehyde 21 (Figure 3A). We treated the UV spot-welded cones with K10PEG5K (N:P ratio of nitrogen in lysine to phosphorus in DNA of 1:0.6, and 2% (v / v) glutaraldehyde). To test the protection against nuclease activity, we placed the samples in DNase I (0.001 U / μl, equivalent to 2.6-fold the typical blood concentration of DNase I). We analyzed the digestion products using negative staining TEM for direct imaging. When uncoated, the cone assembly was completely digested after 8 hours of incubation with DNase I, while when oligolysine-PEG coated and cross-linked with glutaraldehyde, the cones remained stable for up to 48 hours with no obvious structural damage (Figure 3E).

[0194] For the intended application of flattening and closing the surface of viral particles, the inward-facing surface of the cone must be functionalized with additional virus-binding moieties. To this end, we introduced single-stranded DNA protrusions (termed "stalks") on the inner surface of the wedge subunits, which can hybridize with sequence-complementary oligonucleotides modified with the selected virus-binding moiety. The position and number of stalks displayed on the wedge surface can be controlled by design. When using strong virus binders such as antibodies, a fairly low density of stalks may be sufficient to capture the virus (Figure 4A); while weak and more broadly binding virus binders such as heparan sulfate (HS) polymers 22 ) may benefit from a higher density of stalks to exploit multivalent and avidity effects. To covalently conjugate the DNA strands to the virus binders, we used sulfo-SMCC linker (sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) for antibody conjugation 11 , and a copper-free click chemistry method for HS derivatives. 12

[0195] With the stabilization and functionalization of the cone assemblies, we tested the ability of the cones to assemble around influenza A / Puerto Rico / 8 / 1934 virus. Hemagglutinin (HA) and neuraminidase (NA) are the two most abundant proteins on the surface of influenza A virus particles. We selected an antibody (see Materials and Methods) that targets a conserved epitope in the HA trimer stem region as the virus-binding moiety and used it at a calculated density of six replicates per wedge subunit. As we observed by direct TEM imaging, the antibody-functionalized cones successfully assembled around influenza virus particles (Figure 4B). The cones adapted and conformed to the shape of influenza particles of various shapes (Figure 4C). To obtain more detailed information about the 3D surface coverage of the selected cone-virus assemblies, we performed negative stain electron microscopy tomography (Figure 4D). Slices of exemplary 3D tomographic images revealed influenza virus particles surrounded by two cones in a sandwich-like assembly. Cones without antibody were not associated with influenza virus (Figure 17).

[0196] To further increase the surface area occluded on the virus particle, we designed a spiked cone assembly in which a second wedge subunit (t2) assembles on the base of the cone (Figure 5A, Figure 5B). The t2 wedge has an oblique angle of 45° and binds to the edge of the t1 wedge through a second set of shape-complementary protrusion and depression patterns (Figure 5A, Figures 18 to 20 for cryo-EM validation, Figures S21 to S22 for assembly characterization). After adding the t2 building block, the total cavity depth and diameter of a single spiked assembly are approximately 125 nm (Figure 5B). Thus, in principle, a single replica of the spiked vertebral body is large enough to completely engulf an influenza virus. Figure 5C shows an exemplary negative stain TEM micrograph of the spiked vertebral body influenza assembly that we obtained. These images reveal the flexibility of the spiked vertebral body and the different configurations it can adopt. The t2 subunit also binds a stalk position on its inner surface for placement of the virus-binding moiety. Similar to the cone assembly, when functionalized with an antibody, the spiked cone variant also successfully formed a complex with influenza A / Puerto Rico / 8 / 1934, as we saw by TEM imaging (Figure 5D). Now, a single replica of the spiked vertebral body is sufficient to completely enclose entire virus particles of different sizes. Negative stain TEM tomography was again used to obtain detailed 3D information. Figure 5E shows a tomographic slice of a 3D tomographic image acquired through an exemplary spiked vertebral body influenza assembly, clearly revealing the influenza virus "guest" deep within the cavity of the spiked vertebral body "host".

[0197] To illustrate the modular functionalization of the viral binding part, we used a more extensively binding heparan sulfate (HS) derivative as an inner coating to capture different virus particles with the spike cone assembly. When 12 replicates of HS were used per wedge subunit, chikungunya, SARS-CoV-2, and Zika virus-like particles (VLPs) were also successfully captured in the spike cone assembly, as determined by direct imaging with negative stain TEM (Figure 23). Depending on the hardness of the virus particle, the host cone or the guest virus particle adapts to each other. For example, Zika virus particles flatten completely upon attachment to the cone, while the cone deforms to match the curvature of the rather spherical and significantly harder chikungunya particles.

[0198] Conclusion

[0199] We present conical DNA origami higher-order assemblies that are formed efficiently and in high yield from a single structural unit. Compared to our previous prototypes, which required weeks to assemble, used multiple structural units, and had low yields (<50%), we achieved a more than 80% increase in assembly yield in a one-pot reaction mixture in 72 hours. We developed the cone assembly mainly for capturing and engulfing large polymorphic virus particles. For this purpose, we demonstrated modular functionalization with user-defined viral binding parts. In one example, we used antibodies to engulf influenza virus with the cones, with up to 60 antibodies displayed per cone. In another example, we used heparan sulfate to capture Zika virus, chikungunya, and SARS-CoV-2 VLPs, with up to 120 HS polymer replicates displayed per cone using the cone assembly. The cone assembly can deform and adapt to the shape of the captured virus particle, as seen here with the polymorphic influenza virus sample, which is advantageous for the targeted applications we envision. We also established post-assembly stabilization treatments for the cones, enabling them to persist in low-salt environments and survive nuclease attack for at least 48 hours. In principle, all DNA components required for our cones can be produced on a large scale by biotechnology 23 . Thus, this study helps to lay the foundation for testing the therapeutic potential of DNA nanostructures that engulf large viruses in vivo. In addition to capturing large viruses, the cone assembly or its variants can also be used for artificial light-harvesting antenna complexes 24,25 , and as candidate structures placed on the surface of nanostructures. 26,27

[0200] Materials and Methods

[0201] The staple strands for the DNA origami reaction were purchased from Integrated DNA Technologies (IDT) and used with standard desalting purification. The SH-modified handle strands were purchased from Biomers at HPLC grade. The PEG-polylysine coating was purchased from Alamanda Polymers. Chikungunya VLPs were purchased from The Native Antigen Company, SARS-CoV-2 VLPs were purchased from Creative Biolabs, Zika VLPs were purchased from Creative Biostructure, and inactivated influenza A / PR / 8 / 34 virus was purchased from Charles River Laboratories.

[0202] DNA Origami Design

[0203] The cross-sections of the triangular structural units t1 and t2 are both arranged in a 3x6 pattern in the square lattice of the DNA helix.

[0204] The DNA origami design of the isosceles triangles t1 and t2 involves corners with different angles as well as the bevel angle. Schematic representations of the important parameters can be found in Figures 6, A and B. To create corners in the DNA origami object, specific deletions need to be made according to the desired angle. The length difference (Δa) between two DNA double helices depends on the angle (α) and the distance (x) between the two helices (x) in Equation (1) below.

[0205]

[0206] x = n*d (2)

[0207] The distance between two helices (x) is the diameter of the DNA double helix (d). The effective diameter of the DNA double helix is 2.1 nm 39 , but considering that in the DNA origami structure, the helices are not closely packed due to electrostatic repulsion, the average value of d is 2.6 nm 40 . Depending on the position (n) of each helix, x varies, and Δa must be recalculated using Equations (1) and (2). In the case of these design parameters, the closer the DNA helix is to the center, the shorter it is.

[0208] The isosceles triangle has two different angles (α and β), and thus two different corner designs are required. The length differences of the helices at these corners will be different (Δa and Δb), and they need to be calculated separately using Equations (1) and (2). The length of any helix (a x or b x ) can be obtained by subtracting from the reference helix (a 0 or b 0) is calculated by subtracting Δa / b from the length. Additionally, a helical rise of 0.34 nm / bp can be used to convert the length of the DNA helix from base pairs to nanometers.

[0209] a x = a 0 -2Δa(x) (3.1)

[0210] b x = b 0 -Δa(x)-Δb(x) (3.2)

[0211] For the corner design, it is important to understand the double helix direction of the DNA strands at the incision position. To reach the other side of the incision, the DNA strand facing the outside of the corner requires a single-stranded segment (Figure 7C). When the staple strand (yellow) faces the outside of the incision, we assign it 5 thymine single-stranded bases, while when it is the backbone (blue), we only assign it one single-stranded base.

[0212] To obtain an assembly with curvature, the sides of the triangle need to be tilted at a certain bevel angle. Figure 7D shows a schematic representation of how the corner design with a certain bevel angle looks. By rotating each DNA helix by a certain angle θ, the original coordinates of the helix (n,m) change from x 0,nm and y 0,nm to x nm and y nm (Figure 7A). The new coordinates can be calculated using the two-dimensional rotation matrix (4). All the triangles in this work are involved such that the three edges always have the same bevel angle, only with different lengths.

[0213] Having

[0214] The length differences required to achieve the desired bevel angle can be calculated using (5.1) and (5.2):

[0215]

[0216] If the bevel angle is designed to be obvious, then the final assembly will form a deep cavity at the cost of a smaller cone diameter; while if it is not so prominent, the depth of the product will become shallower, but the diameter will become larger.

[0217] Table 2 summarizes the actual values of the corner angles (α and β), the bevel angle (θ), and the reference helix lengths (a x and b x ).

[0218] Table 4. Design parameters of t1 and t2 referring to Figure 7.

[0219] t1 t2 α 74.6° 78.3° β 30.7° 23.5° θ 9.5° 45° <![CDATA[a 0 > 122bp 122bp <![CDATA[b 0 > 232bp 300bp

[0220] Folding of DNA Origami Triangular Subunits:

[0221] Self-assembly ("folding") in a one-pot reaction mixture of DNA origami structures, which contains 50 nM single-stranded scaffold DNA (M13, 8064 bases) and 250 nM of each staple strand in a standardized "folding buffer" (FoB15) at pH 8.00. The folding buffer contains 15 mM MgCl 2 , 5 mM Tris base solution, 1 mM EDTA, and 5 mM NaCl. The production of scaffold M13 was as described previously (Sequence Supplementary Note 1) 28 . The folding reactants were subjected to a thermal annealing ramp (60 °C to 44 °C with a 1 °C / h decrease) in a Tetrad (Bio-Rad) thermal cycler.

[0222] Purification of Triangular Subunits and Self-Assembly of Cones:

[0223] All objects were purified using agarose gel extraction (1.5% agarose, containing 0.5×TBE and 5.5 mM MgCl 2 ) and centrifuged at maximum speed for 60 minutes to remove residual agarose particles. Typical subunit concentrations ranged from 5 nM to 50 nM, while the assembly time was in the range of 3 to 5 days. Incubation at 25 mM MgCl 2 concentration and at 40 °C for at least 72 hours resulted in good cone assembly. Assembly of spiky cones with t2 required 40 mM MgCl 2 and a longer incubation time (about 4 days).

[0224] Stabilization of Cones for In Vivo Applications:

[0225] The assembled cones were UV cross-linked at 310 nm using an Asahi Spectra xenon light source 300W MAX-303 for at least 20 minutes. The cones were incubated with a mixture of K10 oligolysine and K10-PEG5K-oligolysine ester (1:1) at an N / P ratio of 0.6:1 for 1 hour at room temperature, as described previously 20. For chemical cross-linking, an appropriate amount of 50% glutaraldehyde stock solution was added to make the final concentration 2% (v / v), incubated for 1 hour at room temperature, and filtered through a 0.5 ml Zeba spin desalting column (7K MWCO). Dnase I activity assays were performed at 0.001 U / μL (a 2.6-fold increase in blood concentration) and incubated at different time points at 37 °C in 1×PBS buffer containing 10 mM MgCl 2 .

[0226] Production of Recombinant Antibodies:

[0227] Specifically targeting influenza A and B hemagglutinin (HA) 29 The sequences of the heavy variable chain and the λ light variable chain of the broadly reactive monoclonal antibody CR9114 against the stalk region were derived from the RCSB Protein Data Bank 4FQI, modified with appropriate cloning restriction sites and sorted as strings from GeneartTM. The DNA fragments encoding the heavy and light chain variable domains were cloned separately into the pBR322-based human IgG1 expression vectors pAbHC or pAbLC_λ vectors. Sanger sequencing performed by Microsynth Seqlab confirmed the correct cloning. The antibody was expressed in 40 ml of HEK293F Expi cells. At the transfection point, the cells were grown to 2.5 x 106 cells / ml. Transfection was performed using the ThermoFisher ExpiFectamine transfection kit and following the included protocol. 40 μg of DNA (20 μg of heavy chain plasmid, 20 μg of light chain plasmid) was transfected using 107 μl of ExpiFectamineTM. After 16 - 18 hours, 200 μl of Enhancer1 and 2 ml of Enhancer2 were added to the transfected cells. The cells were incubated in a 37 °C, 8% CO2 incubator with shaking at 125 rpm for 5 days to express the antibody. The supernatant was clarified by centrifugation at 1,000 g for 10 minutes and then at 4,000 g for 15 minutes. The clarified supernatant was sterile filtered (0.2 μm microporous sterile filter), and 0.05% NaN 3 was added during storage. The HiTrap rProtein A FF 1 ml column was loaded with the supernatant overnight at 4 °C at a flow rate of 1 ml / min. The column was then washed with 50 ml of PBS to wash away any unbound residues. The antibody was eluted using 0.1 M glycine at pH 3.2 and fractionated 4 times in 2.5 ml. Each fraction was immediately neutralized to a final pH of 7.3 with 1 M Tris / HCl, pH 9. The buffer was exchanged to PBS using a pD10 column. For storage preparation, the antibody was concentrated or diluted to the desired concentration, centrifuged at 14,000 g for 30 minutes, and then sterile filtered (22 μm).

[0228] Antibody conjugation with DNA:

[0229] An oligonucleotide having a sequence complementary to the origami handle (5'-TGCCTAATCTCTACCTACTCTACTGC-3'; SEQ ID NO: 1408) and modified with a thiol group at the 3'-end was conjugated to the antibody anti-HA CR9114 (100 μg) using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate crosslinker. The product was purified using proFIRE (Dynamic Biosensor). The DNA-modified antibody was added to the assembled and UV-welded cones at a 1:1 stoichiometric ratio with respect to the number of handles and incubated for 1 hour at room temperature.

[0230] Heparin sulfate conjugation to DNA:

[0231] The experimental protocol was as previously described by Monferrer et al. 12

[0232] Virus and VLP encapsulation:

[0233] Pre-assembled and UV-welded cones in 1× PBS containing 10 mM MgCl 2 were mixed with virus or VLP samples at an appropriate ratio. The samples were incubated for 2 hours at room temperature. The sample volume for TEM analysis was typically in the range of 5 - 10 μL of the total solution, at a concentration of ~10 nM triangular origami. Negative-stained TEM grids were prepared immediately after 2 hours of incubation.

[0234] Negative-stained TEM:

[0235] Depending on the origami and MgCl 2 concentration, the samples were incubated for 90 to 120 seconds under a glow discharge (45 s, 35 mA) on a forrmvar carbon-coated Cu400 TEM grid (Electron Microscopy Sciences). Next, the grids were stained for 30 seconds with an aqueous solution of uranyl formate 2% containing 25 mM NaOH. Imaging was performed at an FEI Tecnai T12 microscope operating at 120 kV, using a Tietz TEMCAM-F416 camera in SerialEM at a magnification of 10000× to 42000×. TEM micrographs were high-pass filtered to remove long-range staining gradients and the contrast was automatically leveled using Adobe Photoshop. To obtain TEM statistics in an unbiased manner, automated grid montages were acquired. For detailed information on the selected particles, negative-stained EM tomography was used as the visualization technique. Tilt series were made from -30° to +30° and micrographs were acquired in 2° increments. The tilt series were processed using Etomo (IMOD) to obtain tomographic images 30The micrographs were aligned with each other by calculating the cross-correlation of a series of successive tilt images. Then, filtered back-projection was used to generate tomographic images. The Gaussian filter used a cut-off value between 0.25 and 0.5 and had a fall-off of 0.035.

[0236] Negative stain data processing:

[0237] We processed the micrographs in CryoSparc 31 and estimated the contrast transfer function (CTF) using CTFFIND4 32 We combined manual picking and TOPAZ automatic picking 33 and extracted particles composed of different numbers of monomers. We performed multiple rounds of 2D classification on the particles to sort them and create class-average images with a higher signal-to-noise ratio. We evaluated the distribution of the assemblies by assigning particles in some 2D classes and manual inspection. We measured the sizes of different types of assemblies using FIJI based on the 2D class-average image data. 34

[0238] Cryo-grid preparation and cryo-EM image acquisition:

[0239] For the triangular DNA constructs, we vitrified each cryo-EM sample using a Vitrobot Mark IV (Thermo Fisher Scientific). We applied 4 μl of the sample to a glow-discharged C-Flat grid (Protochips) (Table 6) and blotted and plunge-froze it with the following Vitrobot settings: temperature of 22 °C, relative humidity of 100%, blotting time of 2 - 2.5 s, and blotting force of -1. For the vertebral assemblies, we used double blotting, which consisted of: applying 4 μl of the sample, incubating on the grid for 60 s, manual blotting, then a second round of sample application, semi-automatic blotting, and plunge-freezing as described above. We used EPU software (Thermo Fisher Scientific) on a Cs-corrected (CEOS) Titan Krios G2 electron microscope (Thermo Fisher Scientific) operating at 300 kV, with a cumulative dose of and a pixel size of magnification of

[0240] Cryo-EM data processing:

[0241] We mainly processed the cryo-EM data in the Relion 4 software suite 35,36For motion correction of the images and CTF estimation, we used Relion implementation and CTFFIND4 respectively 32 We used TOPAZ to semi-automatically pick up particles 33 、extract particles and remove particles damaged by grid contamination picked up incorrectly through multiple rounds of 2D. Using the low-resolution ab initio initial model created in Relion, we solved the problem of structural heterogeneity through 3D classification and reconstructed the 3D fine map. We applied per-particle motion correction and dose weighting to receive a set of polished particles and reconstructed the 3D fine map at a higher resolution. We post-processed the map using the 0.143 FSC criterion by applying a low-resolution mask and low-pass filtering and sharpening based on Fourier shell correlation (FSC) estimation. For version 1 of triangle 2, we used CryoSparc to reconstruct the final map, including post-processing 31 We used ChimeraX to measure the size of the electron density map in 3D and render the image 37

[0242]

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Claims

1. A three-dimensional polynucleotide-based open shell [1] (Figure 26) that encloses a cavity [2] and includes an opening [3] for accessing the cavity, comprising an n-sided pyramid [4] formed by n identical replicas of an acute isosceles triangular prism t1 [5] of a first type, where n is an integer selected from 7, 8, 9, 10, 11, 12, 13, 14, and 15, where the base plane [6] of each prism points to the outside of the open shell, and the upper plane [7] points to the cavity, where two large side planes [8, 9] of each prism contain a first pattern [10] and a second pattern [11] of one or more protrusions and / or one or more receiving portions, where the first pattern and the second pattern are complementary to each other, and where the small side plane [12] includes a third pattern [13] of one or more protrusions and / or one or more receiving portions; where the acute isosceles triangular prism of the first type is a self-assembling DNA-based structural unit.

2. The three-dimensional polynucleotide-based open shell according to claim 1, further comprising n replicas of an acute isosceles triangular prism t2 [14] of a second type, where the first side [15] of each prism points to the outside of the open shell, and the opposite side [16] points to the cavity and / or to the opening for accessing the cavity, where one plane [17] of the second type of prism structure [14] includes a fourth pattern [18] of one or more protrusions and / or one or more receiving portions that is complementary to the third pattern [13].

3. The three-dimensional polynucleotide-based open shell according to claim 1 or 2, where the self-assembling DNA-based building block contains between 7,500 and 10,500 base pairs, and / or where the molecular weight of each self-assembling DNA-based structural unit is between 4.5 MDa and 7 MDa.

4. The three-dimensional polynucleotide-based open shell according to any one of claims 1 to 3, where the upper plane [7] and / or the opposite side [16] when present contain one or more attachment sites for the attachment of one or more binding molecules.

5. The three-dimensional polynucleotide-based open shell according to claim 4, where the binding molecule is selected from antibodies and antigen-binding fragments thereof and constructs comprising at least one sulfonated or sulfated polysaccharide group.

6. The three-dimensional polynucleotide-based open shell according to claim 5, where the binding molecule is an scFv fragment.

7. The three-dimensional polynucleotide-based open shell according to claim 5, where the binding molecule is a construct comprising one or two sulfonated or sulfated polysaccharide groups.

8. The three-dimensional polynucleotide-based open shell according to claim 7, where the binding molecule is independently selected from the following list: heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate, dextrin 2-sulfate, aptamer, peptide, host-receptor domain, and sialic acid.

9. The three-dimensional polynucleotide-based open shell according to any one of claims 3 to 8, wherein each prism contains between 1 and 45 of said attachment sites.

10. The three-dimensional polynucleotide-based open shell according to claim 9, wherein each prism contains between 3 and 10 attachment sites.

11. The three-dimensional polynucleotide-based open shell according to any one of claims 3 to 10, wherein the attachment site is a first single-stranded oligonucleotide.

12. The three-dimensional polynucleotide-based open shell according to claim 11, wherein the binding molecule is attached to the attachment site by a second single-stranded oligonucleotide that is linked to the binding molecule and is complementary to the first single-stranded oligonucleotide.

13. The three-dimensional polynucleotide-based open shell according to any one of claims 1 to 12, wherein each of the first type of acute isosceles triangular prisms and, if present, the second type of acute isosceles triangular prisms is a DNA-based nanostructure formed from DNA-based building blocks.

14. The three-dimensional polynucleotide-based open shell according to claim 13, wherein the DNA-based nanostructure is formed from a single-stranded DNA template strand and a set of oligonucleotides complementary to the single-stranded DNA template, wherein each of the oligonucleotides is complementary to a continuous segment of DNA sequence on the single-stranded DNA template or to at least two non-continuous segments of DNA sequence on the single-stranded DNA template.

15. The three-dimensional polynucleotide-based open shell according to any one of claims 1 to 14, wherein n is an integer selected from 9, 10, 11, 12, and 13.

16. The three-dimensional polynucleotide-based open shell according to any one of claims 1 to 15, which further comprises chemical cross-linking between different prisms.

17. The three-dimensional polynucleotide-based open shell according to claim 16, wherein the chemical cross-linking is obtained by ultraviolet irradiation.

18. The three-dimensional polynucleotide-based open shell according to any one of claims 1 to 17, which further comprises a coating of the outer surface of the open shell with a polycationic molecule.

19. The three-dimensional polynucleotide-based open shell according to claim 18, wherein the polycationic molecule is polylysine.

20. The three-dimensional polynucleotide-based open shell according to claim 19, wherein the polycationic molecule is polylysine-PEG.

21. The three-dimensional polynucleotide-based open shell according to claim 19 or 20, which further comprises cross-linking of the free amino groups of the polylysine.

22. The three-dimensional polynucleotide-based open shell according to claim 21, wherein the cross-linking is carried out with an alkane dialdehyde.

23. The three-dimensional polynucleotide-based open shell according to claim 22, wherein the cross-linking is carried out with glutaraldehyde.

24. The three-dimensional polynucleotide-based open shell according to any one of claims 1 to 23, which is used for treating a patient infected with a virus, viral particle or subviral particle, suspected of being infected with a virus, viral particle or subviral particle, or at risk of being infected with a virus, viral particle or subviral particle.

25. A composition comprising a mixture of the three-dimensional polynucleotide-based open shells according to any one of claims 1 to 23, wherein the mixture comprises three-dimensional polynucleotide-based open shells having an n value in the range of 7 to 15.

26. The composition according to claim 25, wherein the mixture comprises three-dimensional polynucleotide-based open shells having an n value in the range of 9 to 13, preferably in the range of 9 to 11.

27. The composition according to claim 25 or 26, which is used for treating a patient infected with a virus, viral particle or subviral particle, suspected of being infected with a virus, viral particle or subviral particle, or at risk of being infected with a virus, viral particle or subviral particle.

28. A method for encapsulating a virus, viral particle or subviral particle, which comprises the following steps: providing a three-dimensional polynucleotide-based open shell according to any one of claims 1 to 23 or a composition according to claim 25 or 26; and contacting the three-dimensional polynucleotide-based open shell or the composition with a medium containing or suspected of containing the virus, the viral particle or the subviral particle.

29. A method for treating a patient infected with a virus, viral particle or subviral particle, suspected of being infected with a virus, viral particle or subviral particle, or at risk of being infected with a virus, viral particle or subviral particle, which comprises the following steps: administering to the patient a three-dimensional polynucleotide-based open shell according to any one of claims 1 to 23 or a composition according to claim 25 or 26.

30. A method for treating a patient infected with a virus, viral particle or subviral particle or suspected of being infected with a virus, viral particle or subviral particle, which comprises the following steps: contacting the patient or the body fluid of the patient with a three-dimensional polynucleotide-based open shell according to any one of claims 1 to 23 or a composition according to claim 25 or 26.

31. A composition comprising a virus, viral particle or subviral particle encapsulated by a three-dimensional polynucleotide-based open shell according to any one of claims 1 to 23 or by a three-dimensional polynucleotide-based open shell from a composition according to claim 25 or 26.

Images