DNA origami traps for large viruses

Abstract

The present invention relates to three-dimensional polynucleotide-based open shells for encapsulating viruses or viral particles, compositions comprising mixtures of such three-dimensional polynucleotide-based open shells, compositions comprising viruses or viral particles encapsulated by such three-dimensional polynucleotide-based open shells, and methods for encapsulating viruses, viral particles, or subviral particles by using such three-dimensional polynucleotide-based open shells.

Description

[Technical Field]

[0001] The present invention relates to three-dimensional polynucleotide-based open shells for encapsulating viruses, viral particles, or subviral particles; compositions comprising mixtures of such three-dimensional polynucleotide-based open shells; compositions comprising viruses, viral particles, or subviral particles encapsulated by such three-dimensional polynucleotide-based open shells; and methods for encapsulating viruses, viral particles, or subviral particles by using such three-dimensional polynucleotide-based open shells. [Background technology]

[0002] Viral infections cause millions of deaths worldwide every year, resulting in immense suffering and morbidity, and place a tremendous burden on societies and economies through other burdens that are not easily measured, such as healthcare costs, lost working hours, and mental health problems associated with the loss or stigma of parents, children, or caregivers. Climate change and global migration are predicted to increase the threat of viral outbreaks as the virus spreads to areas that were previously too cold for vectors to survive. The burden of viral infections will be further exacerbated by human encroachment on habitats, urbanization and megacities with increasing population density, increased movement both locally and long-distance, and many other disease emergence factors. 41 Viruses are the class of pathogens most likely to adapt to new environmental conditions due to their short generation time and genetic variability that allows for rapid evolution. 42For the majority of viral diseases (approximately 70% of viruses currently registered with the WHO), there is no effective treatment. Almost all existing antiviral therapies target specific viruses and are not applicable to newly emerging pathogens. In addition, antiviral therapies typically face the challenge of needing to be started immediately after infection, before the viral load becomes too high to cause disease symptoms, in order to be effective. While emerging viral threats require a rapid response, there are no widely applicable, readily available antiviral drugs.

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

[0004] Viruses come in various shapes and sizes. Their dimensions range from the 10 - 1000 nm scale. For example, adeno - associated virus (AAV) is a fairly small icosahedral non - enveloped virus with a reproducible diameter of approximately 20 nm per particle. Influenza virus is a medium - sized virus with an envelope, and its dimensions are on the 80 - 150 nm scale. Influenza virus is also polymorphic, that is, the particles can take on various shapes and sizes including spherical, peanut - shaped, or even filamentous. Mimivirus is representative of a fairly large virus with a diameter of about 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 function on a molecule - to - molecule basis, which means that one copy of the antiviral agent targets one viral surface protein. More recently, two - dimensional 8~10 and three - dimensional 11,12As exemplified by DNA architectures, a multivalent antiviral concept is presented that shows multiple virus binding molecules in a complex geometric structure intended to match the mesoscale structural aspects of a target pathogen. Multivalent virus-coated nanoarchitectures offer an additional option for harnessing the avidity effect associated with multivalent interactions between antiviral agents and viruses. Multivalent binding results in an exponential amplification of binding strength with valency, enabling the achievement of virtually irreversible target binding with individually weak and reversible virus binders. Thus, for example, changes in the virus surface that reduce the binding strength of individual binders caused by mutational drift may be less of a problem in the context of multivalent antiviral agents compared to monovalent binders. Also, the virus binding moieties used in the multivalent nanoarchitecture itself are not necessarily required to have neutralizing activity since the entry inhibition effect will be at least partially achieved by the virus surface blocking material of the DNA nanoarchitecture.

[0007] Icosahedral DNA origami half-shell 11 has previously been found to be able to neutralize viruses up to 85 nm in diameter by mechanically blocking the binding interaction with the cell surface, and thus prevent infection of host cells. For example, there is a need to extend that approach so that such pathogens can also be targeted, as there are numerous highly relevant large human virus pathogens such as influenza virus, coronavirus, or herpes virus. Influenza virus is an enveloped virus with dimensions on the 80 - 200 nm scale that occurs in various shapes including spherical, peanut-shaped, and filamentous. 13 However, previously developed prototypes of shells for encapsulating viruses were either too limited in size and shape to accommodate such virus particles or too cumbersome to produce for practical use.

[0008] Viral genomes mutate frequently, which can reduce the success rate of treatment options such as vaccination, or even lead to their disappearance. Therefore, there is a strong need for therapeutic interventions that can rapidly adapt to newly emerging developments regarding the infectivity of a given virus. None of the above approaches possess sufficient modularity and flexibility to rapidly adapt their structure to viral mutations.

[0009] Therefore, although various strategies for treating viral infections have been developed or proposed, there is still a need to develop a concept for a general-purpose antiviral drug platform that targets various viral pathogens. Specifically, a concept that does not rely on detailed prior knowledge of the genetics and characteristics of the target virus would be desirable. In addition, it is particularly important to develop an antiviral drug platform suitable for mass production. [Overview of the project] [Problems that the invention aims to solve]

[0010] The object of the present invention is to provide a construct that enables the encapsulation of viruses, viral particles, or subviral particles. A solution to this problem, namely the use of simple polymeric components such as DNA-based nanostructures, has not yet been taught or suggested by the prior art. [Means for solving the problem]

[0011] Accordingly, in one embodiment, the present disclosure comprises a three-dimensional polynucleotide-based open shell [1] (Figure 26) enclosing a cavity [2] and including an opening [3] for accessing the cavity, comprising an n-pyramid [4] formed by n identical copies of a first type acute isosceles frustum t1 [5], where n is an integer selected from 7, 8, 9, 10, 11, 12, 13, 14, and 15, where the base [6] of each frustum points to the outside of the open shell, the top [7] points to the cavity, and the two large The present invention provides a three-dimensional polynucleotide-based open shell in which a large side [8, 9] comprises a first pattern

[10] and a second pattern

[11] of one or more protrusions and / or one or more receptors, the first and second patterns being complementary to each other, and a small side

[12] comprises a third pattern

[13] of one or more protrusions and / or one or more receptors, and the first type of acute isosceles triangular frustum is a self-assembling DNA-based component containing 7,500 to 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 the treatment of patients who are 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 compositions according to the present invention for use in the treatment of patients who are infected with, suspected of being infected with, or at risk of being infected with a virus, viral particles, or subviral particles.

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

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

[0017] In another aspect, the present invention relates to a method for treating a patient who is infected with or suspected to be infected with a virus, viral particle, or subviral particle, the method comprising the step of bringing the patient or the patient's bodily fluids into contact with a three-dimensional polynucleotide-based open shell or a composition according to the present invention.

[0018] In another aspect, the Disclosure provides compositions comprising viruses, viral particles, or subviral particles 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] This disclosure intends to include all combinations of any one or more of the embodiments described above, as well as any one or more of the embodiments described in the Detailed Description and Examples.

[0020] Other features, purposes, and advantages of the compositions and methods described herein will become apparent from the description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0021] [Figure 1] The following diagrams show n=10 C10 cone DNA origami designs (n-pyramids as defined in claim 1.a). (A) Left: Schematic model of the C10 cone shell assembly. The cylinder represents a single DNA double helix. Each cone is designed to contain 10 isosceles triangular subunits. Right: Schematic diagram of the C10 cone covering a virus particle. (B) Schematic model of the subunit design implemented in multilayer DNA origami with a square lattice packing. Arrows indicate shape-complementary docking sides located on sides 1 and 2 (S1 and S2). (C) 3D electron density maps determined by single-particle cryo-electron microscopy revealing the close agreement between the designed shape of the wedge subunits and the actual overall shape (see Figure 9 for average values ​​and field micrographs of the cryoEM 3D class).

[0022] [Figure 2] Characterization of conical assemblies is shown. (A) Laser scan fluorescence image of a 1% agarose gel obtained by electrophoresis of the conical assembly reaction using a sample taken at the indicated time. The wedge subunit concentration was 5 nM, the incubation temperature was 40°C, and the solution contained 25 mM MgCl2. M: Marker lane. Sc: M13-8064 scaffold (for reference). (B) Exemplary negative stained TEM micrograph showing the field containing the conical assembly product. Inset: Schematic diagram of a typical orientation in which the cone adheres to the TEM support grid. Scale bar: 100 nm. (C) Two-dimensional TEM class mean (1) of distinct conical assembly species with base attachment orientation. Scale bar: 50 nm. (D) (1) 2D class mean inner diameter measurements of each cone, as well as their occurrence frequency. (E) Cryogenic EM field micrograph showing different cone orientations. Scale bar: 100 nM. (F) Cryo-EM 3D reconstruction of C9 and C10 cones based on internal diameter and depth measurements.

[0023] [Figure 3]This diagram illustrates the stabilization of cone assemblies for future in vivo applications. (A) Schematic diagram of the stabilization workflow: UV point welding, oligolyzin-PEG coating, and glutaraldehyde crosslinking of the coating. (B) Schematic design diagram showing details of the wedge subunit chain diagram indicating the position of additional thymidine (yellow dots) for UV point welding of the t1 subunit. The diagram was created using caDNAno v0.2.4.38. Blue: scaffolding chain, gray: staple chain. (C) Laser scan fluorescence image of a 1% agarose gel electrophoresed with the cone assembly reaction after exposure to irradiation with 310 nm light for the indicated time. The gel was electrophoresed in 3 mM MgCl2. This is a condition under which uncrosslinked cones immediately decompose into wedge subunits (see control or 0 min lane, e.g.). Inset: Magnified view of the high molecular weight circular cone assembly product. Each band is attributed to a closed cone with the indicated number of wedge subunits. (D) Exemplary negative staining TEM image of an irradiated cone versus an unirradiated (and therefore unstabilized) cone in the presence of the indicated MgCl2 concentration. Scale bar: 100 nm. (E) Exemplary negative staining TEM image of a single UV point-welded cone assembly treated with DNase I (0.001 U / μL) compared to a sample additionally coated with oligolyzin-PEG (1:0.6, P:N ratio) and chemically crosslinked with glutaraldehyde. Scale bar: 100 nm. In this context, with respect to Figures 3B, 24 and 25 (see below), it should be noted that these figures show schematic diagrams of some of the complex arrangements of different oligonucleotides that form the polynucleotide-based open shells of the present invention. All oligonucleotides used to form these polynucleotide-based open shells are listed in Tables 1-3 and included in the sequence listings. Tables 1-3 thus contain all the sequence information necessary to generate the nanostructures schematically shown in Figures 3B, 24 and 25, and these are included for illustrative purposes only. These figures do not contain any additional sequence information.

[0024] [Figure 4](A) Schematic diagram showing how cones can function at the virus binding site. Red: Single-stranded DNA extension called the "handle". Blue: DNA-tagged antibody. (B) Capture of influenza A / PR / 8 / 34 virus by a cone assembly featuring 6 copies of CR9114 antibody per wedge subunit. Negative stained TEM images of a single virus particle covered with different numbers of cones. Depending on the size and overall shape of the virus particle, up to 3 cones coordinated to cover the entire spherical / peanut-shaped virus, and more cone copies adapted to cover fibrous influenza particles. Scale bar: 50 nm. (C) Negative stained TEM images of cones adjusted to capture multiple virus particles at once. Scale bar: 50 nm. (D) Slices across single-particle 3D tomography of influenza virus completely encapsulated by two cones in a sandwich-like assembly, acquired in a negative stained TEM tilted series. Scale bar: 25 nm.

[0025] [Figure 5] The image shows a spiked cone assembly with improved surface coverage. (A, B) A schematic model of the spiked cone design utilizing a second wedge block (t2) designed to assemble at the base of the cone. (C) Exemplary negative-stained TEM micrographs of the spiked cone assembly from different distinct viewpoints. (D) Exemplary TEM micrograph showing an influenza A / PR / 8 / 34 virus particle encapsulated in a spiked cone assembly functionalized with 6 × CR9114 antibody per wedge subunit. (E) A slice of negative-stained 3D TEM tomography of a single influenza virus particle completely encapsulated by a single spiked cone, achieving better surface coverage than a non-spiked cone. All scale bars: 50 nm.

[0026] [Figure 6]Schematic diagrams of the design parameters for t1 and t2 are shown. (A) Cross-sectional view of a 3×6 DNA helix in a square grid arrangement, in both linear and inclined configurations. (B) Representation of corner angles (α and β) and reference helix lengths (ax and bx). (C) Representation of single-stranded DNA loops bridging the corner design. (D) Representation of the beveled corner design.

[0027] [Figure 7] This shows the Cryo-EM determination for t1 version 1. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class average. (C) 3D histogram showing particle orientation distribution. (D) FSC plot. (E) Electron density maps from six different viewpoints. Scale bar: 25 nm.

[0028] [Figure 8] Cryo-EM measurements of t1 version 2 are shown. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class average. (C) 3D histogram showing particle orientation distribution. (D) FSC plot. (E) Electron density maps from six different viewpoints. Scale bar: 25 nm.

[0029] [Figure 9] This shows the Cryo-EM determination for t1 version 3. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class average. (C) 3D histogram showing particle orientation distribution. (D) FSC plot. (E) Electron density maps from six different viewpoints. Scale bar: 25 nm.

[0030] [Figure 10] The Cryo-EM electron density maps of triangles t1 and t2 are shown. The DNA origami design was validated using a repeating process with Cryo-EM. This allowed us to correct the twist in the initial version, resulting in a nearly twist-free object (the final version).

[0031] [Figure 11]Negative staining TEM of the t1 folding reaction crude is shown. This micrograph shows how the t1 triangle begins to assemble into a cone during the folding reaction. Excess staples from the folding are observed in the background. Scale bar: 100 nm.

[0032] [Figure 12] Negative staining TEM of nonspecific cone stacking induced by high ionic intensity is shown. Side and top views of nonspecific cone stacking. Scale bar: 100 nm.

[0033] [Figure 13] This shows the 2D class average of cones extracted from negative stained TEM. Cones attached at the apex have a larger diameter and frayed circumference compared to cones attached at the base, which contain the same number of wedge components. Scale bar: 100 nm.

[0034] [Figure 14] Cryo-EM of cones. (A) Electron density map of a C9 cone from a different viewpoint. Scale bar: 50 nm. (B) Electron density map of a C10 cone from a different viewpoint. Scale bar: 50 nm. (C) 3D histogram showing the orientation distribution of a C9 cone. (D) The same as C for a C10 cone.

[0035] [Figure 15] The images show 3D measurements of the dimensions of the cryo-EM reconstruction. (A) C9 cone. (B) C10 cone.

[0036] [Figure 16]This shows a multibody analysis of a C9 object. (A) Nine masks (colored, semi-transparent) surrounding the reconstruction of the C9 object used for multibody refinement. (B) Principal component analysis of the refined orientation of individual rigid bodies from the nine-body multibody refinement. (C) Distribution of particle weights along the first principal component (PC). (D) Reconstruction of two subsets of the particle population. 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.

[0037] [Figure 17] This image shows a negative stained TEM of a negative control for influenza A / PR / 8 / 34 capture by a cone. The field of view shows no binding of influenza virus particles without antibody coating. Scale bar: 100 nm.

[0038] [Figure 18-1] Cryo-EM determination of t2 version 1 is shown. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class average. (C) Histogram showing particle orientation distribution. (D) FSC plot. (E) Electron density maps from six different viewpoints. Scale bar: 25 nm.

[0039] [Figure 18-2] Cryo-EM determination of t2 version 1 is shown. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class average. (C) Histogram showing particle orientation distribution. (D) FSC plot. (E) Electron density maps from six different viewpoints. Scale bar: 25 nm.

[0040] [Figure 19] This shows the Cryo-EM determination for t2 version 2. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class average. (C) 3D histogram showing particle orientation distribution. (D) FSC plot. (E) Electron density maps from six different viewpoints. Scale bar: 25 nm.

[0041] [Figure 20] This shows the Cryo-EM determination for t2 version 3. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class average. (C) 3D histogram showing particle orientation distribution. (D) FSC plot. (E) Electron density maps from six different viewpoints. Scale bar: 25 nm.

[0042] [Figure 21] The cylindrical representation of the assembly features of triangles 1 and 2 is shown. (A) Side 3 of t1 can be functionalized with a projection perpendicular to sides 1 and 2 for the assembly of t2, which has complementary features in the form of a recess. (B) Dimeric representation from two different viewpoints.

[0043] [Figure 22] Characterization of the t1-t2 dimer assembly is shown. (A) Exemplary laser-scanned fluorescence image of a 1.5% agarose gel showing assembly of t1 and t2 in a 1:1 ratio over two days. t1 and t2 were incubated at 40°C in the presence of 25 mM MgCl2 with a triangular monomer concentration of 5 nM. Sc:M13-8064 scaffold (for reference). Sides 1 and 2 of t1 were passivated to avoid conical assembly. (B) Percentage (%) of fully assembled dimers at different time points and different MgCl2 concentrations. Percentages (%) were extracted from agarose gels as shown in (A). Error bars indicate the standard deviation of the three scans.

[0044] [Figure 23]This demonstrates broadband virus capture by heparan sulfate-modified spiked cones. (A) Schematic diagram showing how the cones can be functionalized at the virus binding site. Red: Single-stranded DNA extension called the "handle". Orange: HS polymer. Capture was performed using spiked cones featuring 12 heparan sulfate moieties per wedge subunit. (B) Exemplary negative-stained TEM micrographs showing captured SARS-CoV-2 and Zika virus-like particles (VLPs). (C) Negative-stained TEM micrographs showing captured Chikungunya VLPs. Due to the small size of CHIK-VLPs, up to three virus particles fit into the large cavity of the spiked cone, and the spiked cone was significantly deformed to maximize contact with the virus. All scale bars: 50 nm.

[0045] [Figure 24] Triangle 1 shows caDNAno blueprints for (A) version 1, (B) version 2, and (C) version 3. Blue: scaffolding chain, colored: staple chain. Designs created with caDNAno v0.2.4.

[0046] [Figure 25] The caDNAno blueprints for (A) version 1, (B) version 2, and (C) version 3 of triangle 2 are shown. Blue: scaffolding chain, colored: staple chain. Designs created with caDNAno v0.2.4.

[0047] [Figure 26] A schematic diagram of the three-dimensional polynucleotide-based open shell of the present invention is shown, including the reference numbers used in the claims. [Modes for carrying out the invention]

[0048] This disclosure provides a construct that enables the encapsulation of viruses, viral particles, or subviral particles.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0050] The terms “comprising” and “including” are used herein in their open-ended and non-restrictive sense, except where specifically noted. Therefore, with respect to such latter embodiments, the term “comprising” includes the narrower term “consisting of.”

[0051] In the context describing this invention (particularly in the context of the following claims), the terms “a,” “an,” and “the,” and similar references, should be interpreted as encompassing both singular and plural forms unless otherwise indicated herein or unless the context clearly contradicts this interpretation. For example, the term “cell” includes multiple cells (including mixtures thereof). When the plural form is used for compounds, salts, etc., it should be interpreted as also meaning a single compound, salt, etc.

[0052] Accordingly, in one embodiment, the present disclosure comprises a three-dimensional polynucleotide-based open shell [1] (see reference numeral 26) enclosing a cavity [2], comprising an n-pyramid [4] formed by n identical copies of a first type acute isosceles frustum t1 [5], where n is an integer selected from 7, 8, 9, 10, 11, 12, 13, 14, and 15, where the base [6] of each frustum points to the outside of the open shell, the top [7] points to the cavity, and the two sides of each frustum The larger side [8, 9] comprises a first pattern

[10] and a second pattern

[11] of one or more protrusions and / or one or more receptors, wherein the first and second patterns are complementary to each other, and the smaller side

[12] comprises a third pattern

[13] of one or more protrusions and / or one or more receptors, wherein the first type of acute isosceles triangular frustum provides a three-dimensional polynucleotide-based open shell in which the self-assembling DNA-based component is a three-dimensional polynucleotide-based open shell.

[0053] In certain embodiments, the self-assembling DNA-based component comprises 7,500 to 10,500 base pairs.

[0054] In certain embodiments, the molecular weight of each self-assembling DNA-based component is between 4.5 and 7 MDa.

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

[0056] In the context of this disclosure, the term “DNA-based, polynucleotide-based open shell” refers to a DNA-based nanostructure formed by a set of DNA-based polymers. DNA-based nanostructures similar to those used in this invention are described in detail in WO2021 / 165528 and Sigl et al., loc. cit.

[0057] In the context of this disclosure, the term "DNA" refers to deoxyribonucleic acid, which consists of a single strand of monomeric units called nucleotides, each nucleotide comprising a nitrogen-containing nucleic acid base, a 2-deoxyribose sugar moiety, and a phosphate group, with each nucleotide being linked in a single strand by a phosphate group that links the 5' OH group of the 2-deoxyribose sugar moiety to the 3' OH group of an adjacent 2-deoxyribose sugar moiety. In certain embodiments, the nitrogen-containing nucleic acid base is independently selected from cytosine [C], guanine [G], adenine [A], and thymine [T]. In certain embodiments, one or more nucleic acid bases are non-standard bases, particularly modified adenosine, particularly N6-carbamoylmethyladenine or N6-methyladenine; modified guanine, particularly 7-deazaguanine or 7-methylguanine; modified cytosine, particularly N4-methylcytosine, 5-carboxylcytosine, 5-formylcytosine, 5-glycosylhydroxymethylcytosine, 5-hydroxycytosine, or 5-methylcytosine; modified thymidine, particularly α-glutamylthymidine or α-ptresinylthymine; uracil or a variant thereof, particularly uracil, base J, 5-dihydroxypentauracil; or 5-hydroxymethyldeoxyuracil; and non-standard bases selected from the list of deoxyarchaeosine and 2,6-diaminopurine. A single strand of DNA can interact with a complementary stretch of DNA through the interaction of complementary nucleic acid bases. Cytosine and guanine, as well as adenine and thymine, are complementary to each other by forming two (A / T) and three (G / C) hydrogen bonds between the nucleic acid bases, respectively. Two single strands of DNA can be perfectly complementary to each other, as in the case of genomic DNA, or they can be partially complementary, including situations where one single strand of 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 helix.

[0058] As is well known, DNA has evolved in nature as a carrier of genetic information that codes for proteins. DNA further includes non-coding regions that have regulatory functions. Therefore, any DNA-based application usually depends critically on a particular DNA sequence and, in most cases, is only possible by naming that particular DNA sequence. In contrast, in the context of this invention, such coding and / or regulatory functions play no role and may or may not be present, because the underlying DNA sequence is designed and selected alone so that a desired arrangement of double helix subunits is formed. Thus, in one embodiment, any form of a long single-stranded DNA sequence, whether naturally occurring DNA (such as bacteriophage DNA) or synthetically produced DNA, can be selected as a template to design a set of short single-stranded DNA sequences, each sequence complementary to one or more different parts of the template and thus forming one or more double helix portions. In summary, all such double helix portions created by the interaction of a complete set of short single-stranded DNA sequences with the template then form a desired three-dimensional arrangement. Starting from a given single-strand template array, the design of a set of complementary elements can be set using known techniques, for example, methods described for the synthesis of separate megadalton-scale objects with structurally distinct 3D shapes. 15,40,49-60 Specifically, caDNAno 38 Elastic network guide molecular dynamics simulation 61 Iterative design can be used in combination with this.

[0059] In addition to complementary nucleic acid base interactions of different stretches of single-stranded DNA via hydrogen bonds, stability and specificity are also important. 62The use of protrusions and recesses, either blunt or sticky ends, to increase the interaction between different DNA strands, including interactions between the ends of two double-stranded DNA helices, enables the design and formation of complex DNA-based nanostructures via the shape complementarity of double-helical subunits. Thus, two three-dimensional arrangements formed according to the previous paragraph can interact with each other through interactions between double-helical subunits present on the two three-dimensional arrangements, including specific interactions between the two three-dimensional arrangements having complementary protrusions and recesses (or knobs and holes).

[0060] In certain embodiments, DNA-based nanostructures are formed from self-assembling DNA-based components.

[0061] In certain embodiments, each of the self-assembling DNA-based components is formed by a single-stranded DNA template strand and a set of oligonucleotides complementary to the single-stranded DNA template, each of which oligonucleotides is complementary to either a single contiguous DNA sequence stretch or at least two non-contiguous DNA sequence stretches on the single-stranded DNA template.

[0062] In certain embodiments, the DNA-based nanostructure consists of 4 to 180 such self-assembling DNA-based components.

[0063] In certain embodiments, the single-stranded DNA template is either single-stranded DNA from a filamentous bacteriophage or derived from single-stranded DNA from a filamentous bacteriophage.

[0064] In the context of this invention, the term "fibrous bacteriophage" refers to a type of bacteriophage or bacterial virus that typically contains a circular single-stranded DNA genome and is characterized by its fibrous shape, which infects Gram-negative bacteria. Examples of fibrous phages include Ff phages such as M13, f1, and fd1 phages, as well as Pf1 phages.

[0065] In certain embodiments, the single-stranded DNA template has the sequence described in Sequence ID No. 1 (M13 8064) (see Table 1). In certain other embodiments, the single-stranded DNA template has the sequence of M13 7249 (see Sequence ID No. 2 in WO2021 / 165528).

[0066] In certain embodiments, the single-stranded DNA is circular.

[0067] In the context of the present invention, a single-stranded DNA template "derived from the single-stranded DNA of a filamentous bacteriophage" refers to a naturally occurring DNA construct of the exposed DNA sequence of a filamentous bacteriophage by one or more of the following: (i) opening of a 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. Any such alteration may have harmful, or at least rather unpredictable, effects on bacteriophage biology, its infectivity and its transmissibility, but such effects play no role in the context of the present invention, for as already stated above, the single-stranded DNA template is used only as a bare template without needing to have any functional properties, and all structural aspects, such as the precise formation of the three-dimensional shape of the self-assembling DNA-based components, are carried out by the appropriate selection of the complementary set of oligonucleotides.

[0068] In certain embodiments, the single-stranded DNA template has at least 80%, particularly at least 90%, and more specifically at least 95% sequence identity with sequences of naturally occurring or publicly available fibrous bacteriophages, in particular sequences of M13, f1, or fd1 phages, especially sequence number 1 (M13 8064) and M13 7249 (see sequence number 2 in WO2021 / 165528). In this context, it should be noted that the single-stranded DNA template is used solely as a template in the invention, so that the exact sequence does not have a biological role and / or function. Instead, the setup of the polynucleotide-based open-shell three-dimensional structure is achieved essentially by synthesizing a set of oligonucleotides complementary to two or more sequence stretches on the single-stranded DNA template, so any sequence of similar length can be used. The set of complementary oligonucleotides can be designed manually, but caDNAno 37 The bacteriophage sequences listed above are given only as examples, as it is easier to do so using computer programs such as the ones mentioned above.

[0069] In the context of this invention, the term "frustum of an acute isosceles triangular pyramid" means a polyhedron in which all vertices lie in two parallel planes and the two planes are in the form of acute isosceles triangles.

[0070] In a particular embodiment, the present invention relates to a DNA-based nanostructure in which each frustum of a triangular pyramid is formed by m triangular planes, where m is an integer independently selected from 4, 5, 6, 7, and 8, particularly independently selected from 5, 6, and 7, and more specifically, the integer is 6, and each of the three or four sides of the m planes is formed by n parallel stretches of DNA double helices, where n is an integer independently selected from 1, 2, 3, 4, 5, and 6, particularly independently selected from 2, 3, 4, and 5, and more specifically, an integer selected from 3 and 4. Each plane is connected to a plane above and / or a plane beyond it by (i) stacking the interactions between the DNA double helices that form the plane, and (ii) in part by DNA stretching in the single-stranded DNA template and / or the oligonucleotide that forms the DNA-based components that bridge at least two of the planes. The present invention relates to a DNA-based nanostructure in which at least two of three or four lateral trapezoids each include a specific pattern of recesses and / or protrusions formed by missing or additional DNA double helix stretches, due to specific interaction with a complementary pattern on another lateral trapezoid of the self-assembling DNA-based component.

[0071] In certain embodiments, the average length of each stretch of n DNA double helices in the n planes of a frustum of a triangular or frustum of a rectangular pyramid is 80 to 200 base pairs.

[0072] In certain embodiments, the triangular prismoid is a triangular frustum.

[0073] In the context of this invention, the term "triangular frustum" refers to a three-dimensional geometric shape in the form of a triangular pyramid, where the tip of the pyramid is removed and the top is a plane parallel to the base of the pyramid.

[0074] In certain embodiments, for at least some of the self-assembling DNA-based components, the length of at least one side of each of the m planes decreases from the first plane to the mth plane, resulting in a bevel angle θ between the plane perpendicular to the first plane and the trapezoidal plane formed by the m sides (see Figure 6). In certain embodiments, all three trapezoidal planes exhibit a bevel angle.

[0075] In certain embodiments, the Bechel angle is 16° to 26°, particularly 18° to 24°, more specifically 20° to 22°, and most specifically about 20.9°.

[0076] In certain embodiments, the DNA-based nanostructure comprises at least one set of self-assembling DNA-based components, each of the three or four lateral trapezoids comprising a specific pattern of recesses and / or protrusions formed by missing or additional DNA double helix stretches due to specific interaction with a complementary pattern on another lateral trapezoid of the self-assembling DNA-based component.

[0077] In a particular embodiment, the three-dimensional polynucleotide-based open shell further comprises n copies of a second type of acute isosceles frustum t2

[14] , wherein a first side

[15] of each frustum points outward from the open shell, and the opposite side

[16] points outward from the cavity and / or opening for accessing the cavity, and one plane

[17] of the second type of frustum structure

[14] includes a fourth pattern

[18] of one or more projections and / or one or more receptacles that are complementary to the third pattern

[13] .

[0078] In certain embodiments, the DNA-based nanostructure comprises two sets of self-assembling DNA-based components, particularly self-assembling DNA-based components t1 and t2.

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

[0080] In the context of this disclosure, the term “RNA” refers to ribonucleic acid, which consists of a single strand of monomeric units called nucleotides, each nucleotide comprising a nitrogen-containing nucleic acid base, a ribose sugar moiety, and a phosphate group, with each nucleotide being linked in a single strand by a phosphate group linking the 5th OH group of the ribose sugar moiety to the 3' OH group of an adjacent ribose sugar moiety. In certain embodiments, the nitrogen-containing nucleic acid base is independently selected from cytosine [C], guanine [G], adenine [A], and uracil [U]. In certain embodiments, one or more nucleic acid bases are non-standard bases, in particular non-standard bases selected from the list of pseudouridine, ribothymidine, and inosine. Unlike DNA, RNA is almost always in single-stranded form, but the formation of double-stranded forms is possible by the interaction of complementary nucleic acid bases, with cytosine and guanine, and adenine and uracil, being complementary to each other by forming two (A / U) and three (G / C) hydrogen bonds between the nucleic acid bases, respectively. In certain embodiments, the disclosure provides polymer-based nanostructures, which are RNA-based nanostructures.

[0081] In the context of the present invention, the term “cavity” refers to the space enclosed by the DNA-based nanostructure. In certain embodiments, the cavity resembles a sphere from which a spherical segment has been cut, and the cut surface is formed by self-assembling DNA-based components at the boundary of the DNA-based nanostructure. In certain embodiments, the cut surface is a large circle, such that the DNA-based nanostructure is a half-shell.

[0082] In certain embodiments, the upper surface [7] and / or, if present, the opposite side

[16] include one or more attachment sites for the attachment of one or more binding molecules that specifically or nonspecificly interact with viruses, viral particles, or subviral particles.

[0083] In certain embodiments, the one or more binding molecules specifically interact with the virus, the virus particle, or the subvirus particle by binding to and inactivating the virus particle or the subvirus particle.

[0084] In certain embodiments, the binding molecule specifically interacts with a virus, viral particle, or subviral particle. Specifically, the binding molecule is selected from an antibody and its antigen-binding fragment, comprising at least one antigen-binding site of the antibody, specifically at least one VH domain of the antibody, or an antibody, specifically at least one combination of the VH domain and VL domain of an scFv fragment.

[0085] In certain other embodiments, the binding molecule nonspecifically interacts with viruses, viral particles, or subviral particles, in particular constructs comprising at least one sulfonated or sulfated polysaccharide group, in particular constructs comprising one or two sulfonated or sulfated polysaccharide groups, more specifically, the sulfonated or sulfated polysaccharide is independently selected from the list of heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate, dextrin 2-sulfate, aptamers, peptides, host-receptor domains, and sialic acid.

[0086] In the context of this application, the term “viral particle” refers to virus-like particles that resemble the three-dimensional structure of an intact virus that is not biologically active. The term “subviral particle” refers to smaller virus-like particles with fewer or smaller subunits, which can be produced for some viruses by not expressing all and / or some of one or more major viral capsid proteins. These artificial virus particles or subviral particles retain the structure and antigenic properties of their native viruses, including virus-specific molecular patterns and high-density B-cell and T-cell epitopes, and induce potent innate, humoral, and cellular immune responses in animals and humans, respectively. 68 .

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

[0088] In the context of this application, the term "sulfonated or sulfated polysaccharide group" refers to a group comprising a polysaccharide containing at least one sulfated hydroxyl group or at least one sulfonated glycosylamino group.

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

[0090] In certain embodiments, the polysaccharide comprising at least one sulfated hydroxyl group or at least one sulfonated glycosylamino group is independently selected from heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate, and dextrin 2-sulfate.

[0091] In certain embodiments, the polysaccharide comprising at least one sulfated hydroxyl group or at least one sulfonated glycosylamino group consists of 3 to 15 disaccharide units, particularly 4, 5, 6, 7, 8, or 9 units, particularly 4 or 9 monosaccharide units.

[0092] In certain embodiments, the disaccharide unit contains two or three O- and / or N-sulfonic acid groups per disaccharide unit, particularly three O- and / or N-sulfonic acid groups.

[0093] In certain embodiments, the polysaccharide comprising at least one sulfated hydroxyl group or at least one sulfonated glycosylamino group is independently selected from heparin, heparan sulfate, and hybrid heparan sulfate.

[0094] In the context of this invention, the terms “heparin” and “heparan sulfate” both refer to a family of linear sulfated heterogeneous polysaccharides found on cell membranes and in the extracellular matrix as part of heparan sulfate proteoglycans (HSPGs). They consist of repeating 1→4 linked disaccharide units, one monosaccharide being an α-D-glucosamine residue and the other monosaccharide being a uronic acid (or, in salt form, uronate). Heparin is a structurally similar polysaccharide found in mast cells as a component of cerglycine proteoglycans. Heparan sulfate and heparin can be defined as follows: Firstly, in heparin, the uronate is mainly α-L-iduronate, while in heparan sulfate, the uronate is mainly β-D-glucuronate, which is the C-5 epimer of α-L-iduronate. Secondly, the D-glucosamine residue is primarily N-acetylated in heparan sulfate, but N-sulfonated in heparin. Finally, at least 70-80% of heparin consists of the disaccharide L-iduronate 2-O-sulfate α(1→4)D-glucosamine N,6-sulfate in heparan sulfate, while approximately 40-60% of the disaccharide consists of (1→4)D-glucuronate β(1→4)D-glucosamine, which can be either N-acetylated or N-sulfonated. In summary, these structural features make heparin more sulfated and therefore more charged than heparan sulfate. However, the names heparin or heparan sulfate are not as clear as this explanation suggests, and it has become clear that polysaccharides isolated from several organisms appear 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-idulonate; highly sulfonated) and “typical” heparan sulfate structural elements (D-glucuronate; N-acetylated and 6-O-sulfonated).

[0095] Heparan sulfate proteoglycan (HSPG) 63;64These are commonly found on the surface of mammalian cells. Weak interactions between viruses and HSPGs are conserved across viral families and therefore appear to be generally beneficial to the viral life cycle. For example, HSPG-viral interactions may enable infection-enhancing diffusive search for their specific host cell receptors on the cell surface. Interactions between heparan sulfate (HS) and viruses are, for example, HS-decorated dendrimers. 3~5 In the form of virus-capturing coatings for condoms based on this technology, it is already being used for medical purposes. In other studies, it is common to surface-functionalize nanoparticles and polymers with HS derivatives to create virus-binding complexes with antiviral activity. 6,7,65,66 Generally, a high level of polyvalence is required to increase the binding strength between HS nanoparticles and viruses. The reversible nature of binding may lead to undesirable non-binding and release of infectious viruses from the virus-capturing coating, or the need to maintain high concentrations of therapeutic activity. 5 .

[0096] In certain embodiments, the polymer-based nanostructure comprises, on average, 1 to 10 binding molecules, particularly 4 to 10, and especially 4, 5, 6, 7, 8, 9, or 10 binding molecules attached to the internal portion of the cavity formed by the polymer-based nanostructure.

[0097] In certain embodiments, one or more of the self-assembling DNA-based components are linked to a construct comprising at least one sulfonated or sulfated polysaccharide group, particularly one or two sulfonated or sulfated polysaccharide groups, that points to the interior of the cavity.

[0098] In certain embodiments, the three-dimensional polynucleotide-based open shell is a DNA-based nanostructure according to the present invention, and at least one binding molecule is located inside the DNA-based nanostructure and is linked to one of the frustums forming the DNA-based nanostructure so as to point to a cavity formed by the DNA-based nanostructure.

[0099] In certain embodiments, each frustum contains 1 to 45 such attachment sites, particularly 1 to 32, and particularly 3 to 10. In certain embodiments, all frustums contain such attachment sites. In other embodiments, only frustum t1 contains such attachment sites, or only frustum t2 contains such attachment sites.

[0100] In certain embodiments, the binding site is a first single-stranded oligonucleotide.

[0101] In certain embodiments, the binding molecule is attached to the binding site by a second single-stranded oligonucleotide, and the second single-stranded oligonucleotide is linked to one or more binding molecules, which are complementary to the first single-stranded oligonucleotide, or otherwise can perform site-directed interactions with the first single-stranded oligonucleotide. In certain embodiments, 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 certain embodiments, each of the first type and the optionally second type of acute isosceles triangular frustum is a DNA-based nanostructure formed of self-assembling DNA-based components, the DNA-based nanostructure being formed of a single-stranded DNA template strand and a set of oligonucleotides complementary to the single-stranded DNA template, each of which oligonucleotides is complementary to either a single contiguous DNA sequence stretch or at least two non-contiguous DNA sequence stretches on the single-stranded DNA template.

[0103] In certain embodiments, the vertex angles of the acute isosceles triangles forming the opposing faces of the acute isosceles triangular frustum are between 15° and 60°, particularly between 20° and 30°.

[0104] In certain embodiments, n is an integer selected from 9, 10, 11, 12, and 13.

[0105] In certain embodiments, the three-dimensional polynucleotide-based open shell further comprises chemical crosslinks between different frustums, and further comprises one or more crosslinks within one of the frustums and / or between two of the frustums.

[0106] In the context of this invention, the term “crosslinking” refers to any permanent or intermittent linkage within one of the frustums of the triangular pyramids and / or between two of the frustums of the triangular pyramids. Any such linkage may be achieved pre-intervention by linking two of the oligonucleotides used to form the self-assembling DNA-based components before assembly, or post-intervention by chemically or photochemically adding the linkage between different parts of a three-dimensional nanostructure, for example. Permanent linkages may be obtained, for example, by photochemically crosslinking T residues appropriately positioned in the structure under the formation of covalent cyclobutanepyrimidine dimer (CPD) bonds. 19 Discontinuous linkages can be created, for example, by photochemically crosslinking the blunt ends of two double-helix subunits between a 3-cyanovinylcarbazole (cnvK) moiety located at the first blunt end and a thymine residue (T) located at the other blunt end. 67 .

[0107] In certain embodiments, the three-dimensional polynucleotide-based open shell further includes chemical crosslinks between different frustums of triangularity.

[0108] In certain embodiments, the chemical crosslinking is obtained by UV irradiation.

[0109] In certain embodiments, the three-dimensional polynucleotide-based open shell further comprises a coating on the outer surface of the open shell having a polycationic molecule.

[0110] In certain embodiments, the polycationic molecule is polylysine, and more particularly, polylysine-PEG.

[0111] In certain embodiments, the three-dimensional polynucleotide-based open shell further comprises crosslinking with the free amino groups of the polylysine, particularly with alkanedialdehydes, and especially with glutaraldehyde.

[0112] In certain embodiments, the opening [3] has a diameter

[19] of 100 to 200 nm.

[0113] In the context of this invention, the term “diameter” refers to the diameter shown in Figure 26

[19] .

[0114] In certain embodiments, the three-dimensional polynucleotide-based open shell has molecular weights of 30 MDa to 80 MDa (t1 only), particularly 40 MDa to 70 MDa, and 60 MDa to 160 MDa (t1 + t2), particularly 80 MDa to 140 MDa.

[0115] In certain embodiments, the volume of the cavity enclosed by the three-dimensional polynucleotide-based open shell is (nm 3 The units are 80,000 to 200,000, especially 100,000 to 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, the range is 9 to 13, with the maximum value being in the range of 9 to 11.

[0118] In another aspect, the present invention relates to compositions according to the present invention for use in the treatment of patients who are infected with, suspected of being infected with, or at risk of being infected with a virus, viral particles, or subviral particles.

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

[0120] In certain embodiments, the method is for removing the virus, viral particles, or subviral particles from the medium. In certain embodiments, the method is for encapsulating the virus, viral particles, or subviral particles and transporting them.

[0121] In certain embodiments, the method for removing the virus, the viral particles, or the subviral particles is a method for treating a patient who is infected with, suspected of being infected with, or at risk of being infected with the virus, the viral particles, or the subviral particles, and the method comprises the step of administering a three-dimensional polynucleotide-based open shell or a composition according to the present invention to the patient.

[0122] In certain embodiments, a method for treating a patient infected with or suspected of being infected with a virus, viral particles, or subviral particles includes the step of bringing the patient or the patient's bodily fluids into contact with a three-dimensional polynucleotide-based open shell or a composition according to the present invention.

[0123] In another aspect, the Disclosure provides compositions comprising viruses, viral particles, or subviral particles 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 certain embodiments, the composition is formed by a process of removing the virus, viral particles, or subviral particles from a medium containing the virus, viral particles, or subviral particles. In certain other embodiments, the composition is formed by a process of incorporating the virus, viral particles, or subviral particles as cargo into a three-dimensional polynucleotide-based open shell.

[0125] In another embodiment, the Disclosure provides a composition comprising a cargo different from a virus, viral particle, or subviral particle, wherein the cargo, such as a complex polymer, is encapsulated by a three-dimensional polynucleotide-based open shell according to the present invention. In a particular embodiment, the cargo is a cytokine. In a particular embodiment, the cytokine is interleukin-6.

[0126] In yet another embodiment, the Disclosure provides a method for encapsulating a cargo different from a virus, viral particle, or subviral particle, such as a composite polymer, 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 containing or suspected to contain the cargo. In a particular embodiment, the cargo is a cytokine. In a particular embodiment, the cytokine is interleukin-6.

[0127] [Table 1] [Table 2-1] Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 Table 6-1 Table 6-2 Table 6-3 Table 6-4 Table 6-5 Table 6-6 Table 6-7 Table 6-8 Table 6-9 Table 7-1 Table 7-2 Table 7-3 Table 7-4 Table 7-5 Table 7-6 [Table 7-7] [Table 7-8] [Table 7-9]

[0128] For clarity, it is understood that certain features of the Invention described in the context of separate embodiments may also be combined and provided in a single embodiment. Conversely, various features of the Invention described in the context of a single embodiment for the sake of brevity may be provided separately or in any preferred partial combination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention and are disclosed herein as if each and all combinations were individually and explicitly disclosed. In addition, all partial combinations of various embodiments and their elements are also specifically encompassed by the Invention and are disclosed herein as if each and all such partial combinations were individually and explicitly disclosed herein.

[0129] The present invention should not be limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0130] To the extent possible under the respective patent laws, all patents, applications, publications, test methods, literature, and other materials cited herein are incorporated herein by reference.

[0131] The following examples illustrate the invention described above and are not intended to limit the scope of the invention. Other test models known to those skilled in the art can also determine the beneficial effects of the claimed invention. [Examples]

[0132] Introduction A polymer shell or tiling that encloses a virus can, in principle, prevent the virus from entering a cell. Here, we describe the design and assembly of a conical DNA origami higher-order assembly that can enclose and tile the surface of pleomorphic viral samples larger than 100 nm. Using cryo-EM, we determine the structure of the subunits and the complete conical assembly and establish a stabilization process that enables use under in vivo conditions. We use the cones, as an example, to enclose influenza A virus particles, as well as SARS-CoV-2, chikungunya, and Zika virus-like particles. Depending on the relative dimensions of the cone and the viral particle, multiple viral particles can be captured per single cone, and multiple cones can also tile the surface of non-spherical viral particles and adapt to them. The conical assemblies are formed in high yield, require little purification, and are suitable for mass production. This is an important requirement for future practical use, including antiviral agents.

[0133] To overcome the limitations mentioned in the section describing the background of the present invention, the inventors herein describe an efficiently assembled DNA origami-based polymer shell system capable of enclosing pleomorphic viral pathogens with a diameter greater than 100 nm, as exemplified by the influenza A virus. The inventors' design concept considers that wedge-shaped components self-limitingly oligomerize into a cone. Planar finite-size assembly 14 This extension to the previous implementation reduces the complexity of the assembly process by using a minimal number of subunit types. The resulting high-yield assembly makes the conical system suitable for the mass production required for future practical use as an antiviral agent.

[0134] Results and Discussion The inventors' conical assembly is designed to be formed from multiple copies of a wedge-shaped component (t1) (see Supplementary Information for design details). The wedge component can be oligomerized via two distinct, self-complementary edges on opposite faces. Oligomerization of the wedge results in a self-closing circular assembly. Given the designed geometry of the wedge, the cone is expected to have 10 faces (Figures 1A, B). The diameter of the base of the cone consisting of 10 wedges is designed to be approximately 120 nm, so that two copies of the cone are large enough to enclose, for example, an influenza virus particle (approximately 80–200 nm) within a sandwich-like assembly (Figure 1A, right).

[0135] The inventors implemented wedge components in a square lattice helical packing using multilayer DNA origami. 15,16 Objects were assembled using DNA origami methods, and the inventors refined and validated their wedge subunit design in an iterative process using single-particle cryo-electron microscopy (cryo-EM) (Figures 7-9). 3D electron density maps determined for a single wedge particle revealed the designed overall shape and shape-complementary docking features of the triangular components (Figure 1C). The initial wedge design showed significant overall torsional deformation, which the inventors then modified to a nearly torsion-free shape (Figure 10).

[0136] The inventors induced the oligomerization of wedge subunits into cones by increasing the ionic strength of the solution after the wedge components were folded from their constituent DNA staples and scaffolding strands. Oligomerization can also occur simultaneously during the wedge assembly reaction, depending on the ionic conditions used (Figure 11). The inventors monitored the formation of cones in a time-dependent manner by gel electrophoretic mobility analysis (Figure 2A). Here, the appearance and disappearance of bands as electrophoretic mobility decreased reflected the gradual oligomerization of wedge subunits as a function of incubation time. Ultimately, the oligomerized material accumulated in a relatively broad band of low electrophoretic mobility.

[0137] The inventors imaged the final oligomerized product using negative-stain transmission electron microscopy (TEM). Micrographs revealed a predominantly circular structure with a conical appearance, consisting of 9, 10, 11, 12, and rarely 13 copies of wedge subunits, respectively (Figure 2B). The degree of heterogeneity regarding the number of wedges per cone observed in the conical oligomer is likely related to the finite elasticity of the wedge components and their interaction interfaces. These properties can be adjusted as needed, similarly to those previously described for planar ring assemblies. 14 However, for this target application, the distribution of cone products encompassing 9 to 13 wedge-range species appears advantageous for handling pleomorphic virus samples.

[0138] The distribution of cone products observed by TEM partially explains the relatively broad band of products observed in gel electrophoresis analysis. Furthermore, the inventors found that in the presence of high magnesium concentrations, such as those used in gel electrophoresis, the cones tend to stack with each other (Figure 12). This explains the formation of smears and aggregates in high-magnesium gel electrophoresis, as shown in Figure 2A. Under low-magnesium conditions, cone-to-cone stacking was absent, as will be further shown below.

[0139] The inventors observed three main preferred orientations of cones in TEM micrographs (Figure 2B, inset): (1) cones adsorbed at their bases to the surface, (2) cones grounded at their apex, and (3) cones attached to their sides. Cones attached at the apex had a larger diameter and a more frayed circumference compared to cones attached at the base, which contained the same number of wedge components. Presumably, in the apex-attached orientation, the adhesive force flattens the cone, and then the wedges break apart. In the base-attached orientation, the cones remained supported by their bases.

[0140] The inventors calculated two-dimensional (2D) class averages from TEM micrographs. This revealed several classes corresponding to different viewpoints (Figure 13) and different cone types. Figure 2C exemplifies the class averages obtained for base-attached cones, each characterized by 9 to 13 wedge subunits. The inventors measured the diameters from undeformed base-attached cones (1) in each average class, and these were in good agreement with the inventors' expectations (Figure 2D). Thus, the C9 cone type had an average inner diameter of 110 nm. C10 had 126 nm, and the largest C13 type had 147 nm. From the 2D class averages, the relative occurrence frequencies of different cone types were also quantified. The most abundant cone was C10, accounting for 37% of the population, followed by C11 (27%), C9 (20%), C12 (15%), and C13 (1%).

[0141] To obtain 3D information of the assembled product, cryo-EM studies were performed on the cones of self-supporting ice. Exemplary cryo-EM fields of view (Figure 2E) show different orientations of partially and fully assembled cones. 3D reconstructions of the C9 and C10 cone species were determined to confirm the overall 3D cone shape (Figures 2F and 14). Electron density maps of both cone species have an elliptical, undulating base. Ellipticity is more pronounced in the C9 cone map. We measured the lengths of the internal short and long axes, which were 100 nm and 122 nm for the C9 species and 114 nm and 131 nm for the C10 species (Figure 15). The cavities of the C9 cones were 42 nm deep, while the cavities of the C10 cones were shallower (39 nm). Negative staining data and the periphery of the cones attached at the base from cryo-EM reconstructions in solution show good agreement (Table 8). The inventors hypothesize that electrostatic interactions between the cone and the carbon surface of the grid used for negative staining TEM result in a planarization effect, thus making the ring shape more rounded. Additionally, surface interactions at the sample-air interface before plunge freezing may have contributed to the particle deformation observed in the 3D map. Reconstruction of a subset of the particle population from cryo-EM data, as well as multibody refinement and principal component analysis, demonstrates some degree of cone flexibility (Figure 16), which is desirable for the intended application.

[0142] At the salt concentrations present in physiological fluids, higher-order assemblies of DNA origami, as presented in this study, would normally dissociate. 17 Wedge monomers may also be prone to denaturation due to repulsive electrostatic forces within them that have not been adequately screened. The physiological environment may also contain nucleases that can degrade exogenous DNA molecules by catalyzing the hydrolytic cleavage of phosphodiester bonds within the DNA backbone. 18 To sustain the inventors' conical assemblies under in vivo-like conditions, the inventors established a three-step post-assembly stabilization process, schematically shown in Figure 3A. The first step utilizes UV-induced crosslinking of thymidine bases positioned in close proximity within the DNA nanostructure. 19Through irradiation at a wavelength of 310 nm, the double bonds of adjacent pyrimidines undergo a [2+2] ring addition reaction to yield a cyclobutanepyrimidine dimer. To UV crosslink the cone assembly ("UV point welding"), the inventors placed additional unpaired thymidine bases at the helical interface of the inter-wedge subunit interaction sites (yellow dots in Figures 3A and 3B). The inventors tested the effectiveness of UV crosslinking of the cones as a function of exposure time to irradiation with a 310 nm light source (Figure 3C). When properly UV welded, the cones become low Mg 2+ While the control samples remained intact when exposed to the concentration, those that were not irradiated or were insufficiently irradiated rapidly dissociated into constituent wedge subunits (Figure 3C, D). The UV-linked cones now appear as five distinct bands in a low ionic strength gel (3 mM MgCl2).

[0143] To protect the conical assembly from nuclease-mediated degradation, the inventors have developed the aforementioned oligoridine-PEG copolymer-based coating. 20 Next, the glutaraldehyde crosslinking of this coating 21 The following was used (Figure 3A). UV point-welded cones were treated with K10PEG5K (a lysine-to-phosphorus N:P ratio in DNA of 1:0.6, and 2% (v / v) glutaraldehyde). To test protection against nuclease activity, the inventors subjected the samples to DNase I (0.001 U / μl, which corresponds to 2.6 times the typical blood concentration of DNase I). Digestion products were analyzed using direct imaging by negative staining TEM. When uncoated, the cone assemblies were completely digested after 8 hours of incubation with DNase I, but when coated with oligolysin-PEG and crosslinked with glutaraldehyde, the cones remained stable for up to 48 hours without apparent structural damage (Figure 3E).

[0144] For intended applications of tiling and occluding the surface of viral particles, the inward-facing surface of the cone must be functionalized with additional virus-binding moieties. For this purpose, the inventors introduced single-stranded DNA overhangs (referred to as "handles") on the inner surface of a wedge subunit that can hybridize with sequence-complementary oligonucleotides modified with selected virus-binding moieties. The position and number of handles on the wedge surface can be controlled by design. When using potent virus-binding agents such as antibodies, a fairly low density of handles may be sufficient to capture the virus (Figure 4A). However, heparan sulfate (HS) polymer 22 Weak, broad-binding virus conjugates, such as those mentioned above, may benefit from high-density handles to utilize their polyvalent and avidity effects. To covalently bind the DNA strand to the virus conjugate, the inventors used a sulfo-SMCC linker (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) for antibody conjugation. 11 For the HS derivatives, a copper-free click chemical approach was used. 12 .

[0145] Having stabilized and functionalized the cone assemblies, we tested their ability to assemble around the influenza A / Puerto Rico / 8 / 1934 virus. Hemagglutinin (HA) and neuraminidase (NA) are the two most abundant proteins present on the surface of influenza A virus particles. We selected an antibody (see Materials and Methods) that targets a conserved epitope in the stem region of the HA trimer as the virus-binding site and used it at a computational density of 6 copies per wedge subunit. As observed by direct TEM imaging, the antibody-functionalized cones successfully assembled around the influenza virus particles (Figure 4B). The cones adapted and formed around influenza particles of various shapes (Figure 4C). To obtain more detailed information regarding the degree of 3D surface coverage by the selected cone virus assemblies, we performed negative-stained electron microscopy tomography (Figure 4D). Exemplary 3D tomography slices reveal an influenza virus particle surrounded by two cones in a sandwich-like assembly. Cones without antibodies did not associate with the influenza virus (Figure 17).

[0146] To further increase the surface area that would be occluded on the virus particle, the inventors designed a spiked cone assembly in which a second wedge subunit (t2) is assembled at the base of the cone (Figures 5A, B). The t2 wedge has a 45° bevel angle and is coupled to the edge of the t1 wedge via a shape-complementary pattern of protrusions and recesses of the second set (see Figures 5A, 18-20 for cryo-EM verification, and S21-22 for assembly characterization). With the addition of the t2 component, a single spiked cone assembly has an overall cavity depth and diameter of approximately 125 nm (Figure 5B). Thus, a single copy of the spiked cone is, in principle, large enough to completely enclose an influenza virus. Figure 5C shows an exemplary negative-stained TEM micrograph obtained by the inventors of a spiked cone influenza assembly. The image reveals the flexibility and different conformations that the spiked cone can take. The t2 subunit also incorporated a handle position on its inner surface to accommodate the virus binding portion. Similar to the cone assembly, when functionalized with antibody, the spiked cone variant successfully formed a complex with influenza A / Puerto Rico / 8 / 1934, as observed by TEM imaging (Figure 5D). Thus, a single copy of the spiked cone was sufficient to completely enclose an entire range of viral particles of varying sizes. Negative-stained TEM tomography was again used to obtain detailed 3D information. Figure 5E shows tomographic slices obtained from 3D tomography acquired from an exemplary spiked cone influenza assembly, clearly showing that the "guest" influenza virus resides deep within the cavity of the "host" spiked cone.

[0147] To illustrate the modular functionalization by the virus-binding portion, the inventors used a spiked cone assembly to capture different viral particles using a broader-binding heparan sulfate (HS) derivative as an internal coating. When 12 copies of HS were used per wedge subunit, chikungunya, SARS-CoV-2, and Zika virus-like particles (VLPs) were also successfully captured within the spiked cone assembly, as established by direct imaging using negative-stained TEM (Figure 23). Depending on the stiffness of the viral particles, either the host cone or the guest viral particles adapted to each other. For example, Zika particles flattened completely upon attachment to the cone, while the cone, rather spherical, deformed to match the curvature of the clearly more stiff chikungunya particles.

[0148] conclusion The inventors present a conical DNA origami higher-order assembly that can be formed efficiently and in high yield from a single component. Compared to previous prototypes that required several weeks for assembly, needed multiple components, and had poor yields (less than 50%), a significantly improved assembly yield of over 80% was achieved with a one-pot reaction mixture over a 72-hour period. The inventors developed a conical assembly primarily for capturing and encapsulating large pleomorphic viral particles. For this purpose, the inventors demonstrated modular functionalization using user-defined virus-binding moieties. In one example, the inventors used antibodies to encapsulate influenza viruses in a cone, presenting up to 60 antibodies per cone. In another example, the inventors used heparan sulfate to capture Zika, Chikungunya, and SARS-CoV-2 VLPs using a conical assembly that presented up to 120 HS polymer copies per cone. The conical assembly can be deformed and adapted to the shape of the captured viral particle, as observed herein with pleomorphic influenza virus samples. This is advantageous for the target applications envisioned by the inventors. The inventors have also established a post-assembly stabilization process for the cones to survive in low-salt environments and withstand nuclease attack for at least 48 hours. All DNA components required for the inventors' cones can, in principle, be mass-produced biotechnologically. 23 Therefore, this study contributes to building a foundation for testing the therapeutic capabilities of DNA nanoarchitectures that encapsulate large viruses in vivo. In addition to capturing large viruses, the cone assemblies or their variants can be used in artificial light-harvesting antenna complexes. 24,25 It can be used as a candidate structure for placement on a nanostructured surface. 26,27 .

[0149] Materials and methods Staple strands for the origami folding reaction were purchased from Integrated DNA Technologies (IDT) and used with standard desalting purification. SH-modified handle strands were purchased from Biomers in HPLC grade. PEG-polylysine coatings were purchased from Alamanda Polymers. Chikungunya VLPs were purchased from The Native Antigen Company, SARS-CoV-2 VLPs from Creative Biolabs, Zika VLPs from Creative Biostructure, and inactivated influenza A / PR / 8 / 34 virus from Charles River Laboratories.

[0150] DNA Origami Design The cross-sections of both triangular components t1 and t2 were arranged in a 3x6 grid on the square lattice of the DNA helix.

[0151] The DNA origami designs of t1 and t2 isosceles triangles include corners with different angles and bevel angles. Schematic diagrams of the important parameters can be found in Figures 6A and 6B. Creating the corners of the DNA origami object requires specific cuts depending on the angle of interest. The difference in length between two DNA double helices (Δa) depends on the angle (α) and the distance between the two helices (x), according to equation (1).

number

[0152] The distance (x) between the two helices is the diameter (d) of the DNA double helix. The effective diameter of the DNA double helix is ​​2.1 nm. 39 However, considering that the helix is ​​not tightly packed due to electrostatic repulsion in the DNA origami structure, the average of d is 2.6 nm. 40 Since x changes depending on the position (n) of each helix, Δa must be recalculated using equations (1) and (2). Using these design parameters, the DNA helix becomes shorter as it approaches the center.

[0153] An isosceles triangle has two different angles (α and β), and therefore, two different corner designs are required. Since the difference in helical length at such corners is different (Δa and Δb), it must be calculated separately using equations (1) and (2). x or b x The length can be calculated by subtracting Δa / b from the length of the reference helix (a0 or b0). Alternatively, the length of the DNA helix can be converted from base pairs to nanometers using a helical increment of 0.34 nm / bp. a x = a0 - 2Δa(x) (3.1) b x = b0 - Δa(x) - Δb(x) (3.2)

[0154] In corner design, it is important to know the double helix orientation of the DNA strand at the nick location. To reach the opposite side of the nick, the DNA strand facing outward from the corner must have a single-stranded segment (Figure 7C). If the staple strand (yellow) faces outward from the nick, we donate five thymidine single-stranded bases to it, but if it is the scaffolding strand (blue), we donate only one single-stranded base to it.

[0155] To obtain a curved assembly, the sides of the triangles must be beveled at a certain angle. Figure 7D shows a schematic diagram of what the corner design looks like at a specific bevel angle. By rotating each DNA helix by an angle θ, the original coordinates of the helix (n,m) become x 0,nm and y 0,nm from x nm and y nm The coordinates change to (Figure 7A). The new coordinates can be calculated using the two-dimensional rotation matrix (4). All triangles in this study were designed so that all three sides always have the same bevel angle, differing only in length.

number

[0156] The difference in length required to apply the material to achieve the desired bevel angle can be calculated using (5.1) and (5.2).

number

[0157] If the bevel angle is designed to be pronounced, the resulting assembly will feature a deep cavity at the expense of a smaller cone diameter. Conversely, if it is not so pronounced, the product will be shallower but have a larger diameter.

[0158] Corner angles (α and β), bevel angle (θ), and length of the reference helix (a x and b x The actual values ​​of ) are summarized in Table 2. [Table 8]

[0159] Folding of DNA origami triangular subunits: DNA origami structures were self-assembled (folded) in a one-pot reaction mixture containing 50 nM single-stranded scaffold DNA (M13, 8064 bases) and 250 nM staple strands in a standardized "folding buffer" (FoB15) containing 15 mM MgCl2, 5 mM Tris bases, 1 mM EDTA, and 5 mM NaCl at pH 8.00. Scaffold M13 was generated as described above (see Supplementary Note 1 for sequence details). 28 The folding reaction was subjected to a thermal annealing lamp (from 60°C to 44°C, a decrease of 1°C / hour) using a Tetrad (Bio-Rad) thermal cycling apparatus.

[0160] Purification of triangular subunits and self-assembly of cones: All objects were purified using agarose gel extraction (1.5% agarose containing 0.5 × TBE and 5.5 mM MgCl2), and the remaining agarose was centrifuged at maximum speed for 60 minutes to pelletize. Typical subunit concentrations ranged from 5 to 50 nM, and assembly times ranged from 3 to 5 days. Conical assemblies proceeded well with a MgCl2 concentration of 25 mM and incubation at 40°C for at least 72 hours. Assembly of spiked cones with t2 required 40 mM MgCl2 and a longer incubation time (approximately 4 days).

[0161] Cone stabilization for in vivo applications: The assembled cones were UV crosslinked at 310 nm for at least 20 minutes using an Asahi Spectra Xenon light source 300W MAX-303. The cones were incubated with a mixture of K10-oligolyzin and K10-PEG5K-oligolyzin (1:1) and N / P in a 0.6:1 ratio at room temperature for 1 hour, as described above. 20 For chemical crosslinking, an appropriate amount of 50% glutaraldehyde stock was added to a final concentration of 2% (v / v), incubated at room temperature for 1 hour, and filtered through a 0.5 ml Zeba spin desalting column (7K MWCO). DNase I activity assays were performed at 0.001 U / μL (2.6-fold increase from blood concentration) and incubated at 37°C at different time points in 1× PBS buffer containing 10 mM MgCl2.

[0162] Recombinant antibody generation: Influenza A and B hemagglutinin (HA) 29The heavy and lambda variable chain sequences of the broadly reactive monoclonal antibody CR9114, which specifically targets the stem region, were derived from the RCSB Protein Databank 4FQI, modified with suitable restriction sites for cloning, and ordered as strings from Geneart®. DNA fragments encoding the variable domains of the heavy and light chains were cloned into either the pBR322-based human IgG1 expression vector pAbHC or pAbLC_lambda vector, respectively. Correct cloning was confirmed by Sanger sequencing (performed by MicrosynthSeqlab). The antibody was expressed in 40 ml of HEK293F Expi cells. Cells were grown to 2.5 × 10⁶ cells / ml at the time of transfection. Transfection was performed using the ThermoFisher ExpiFectamine transfection kit, following the protocol provided therein. 40 μg of DNA (20 μg of heavy chain plasmid, 20 μg of light chain plasmid) was transfected using 107 μl of ExpiFectamine®. After 16–18 hours, 200 μl of enhancer 1 and 2 ml of enhancer 2 were added to the transfected cells. The cells were incubated in a 125 rpm shaking incubator at 37°C and 8% CO2 for 5 days to express antibodies. The supernatant was clarified by centrifugation at 1,000 g for 10 minutes, followed by 4,000 g for 15 minutes. The clarified supernatant was filtered through a sterile filter (0.2 μm milipore Steritop filter), and 0.05% NaN3 was added for storage. The supernatant was packed into a HiTrap rProtein A FF 1 ml column and incubated overnight at 4°C at a flow rate of 1 ml / min. Next, the column was washed with 50 ml of PBS to remove any unbound residue. The antibody was eluted using 0.1 M glycine (pH 3.2) and fractionated four times in 2.5 ml portions. Each fraction was immediately neutralized with 1 M Tris / HCl (pH 9) to a final pH of 7.3. The buffer was replaced with 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 filtered through sterile filtration (22 μm).

[0163] Conjugation of Antibodies to DNA: An oligonucleotide having a sequence complementary to the origami handle (5’-TGCCTAATCTCTACCTACTCTACTGC-3’; SEQ ID NO: 1408) and with its 3’ end modified with a thiol group was coupled to the anti-HA CR9114 antibody (100 μg) using a sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate crosslinker. The product was purified using proFIRE (Dynamic Biosensors). The DNA-modified antibody was added to the UV-welded cone assembled in a 1:1 stoichiometry with respect to the number of handles and incubated at room temperature for 1 hour.

[0164] Conjugation of Heparan Sulfate to DNA: The experimental protocol was as previously described by Monferrer et al. 12 as described previously.

[0165] Encapsulation of Viruses and VLPs Pre-assembled and UV-welded cones in 1×PBS containing 10 mM MgCl2 were mixed with the virus or VLP sample at an appropriate ratio. The samples were incubated at room temperature for 2 hours. The normal amount of sample for TEM analysis ranges from 5 - 10 μL of the total solution at an approximate triangular origami concentration of 10 nM. Immediately after the 2-hour incubation, negative stain TEM grids were prepared.

[0166] Negative Stain TEM The samples were incubated on a formvar carbon-coated Cu400 TEM grid (Electron Microscopy Sciences) with glow discharge (45 s, 35 mA) for 90 - 120 s depending on the origami and MgCl2 concentration. Next, the grids were stained with a 2% uranyl formate aqueous solution containing 25 mM NaOH for 30 s. Imaging was performed at magnifications of 10,000 - 42,000 in SerialEM using a FEI Tecnai T12 microscope operated at 120 kV with a Tietz TEMCAM-F416 camera. TEM micrographs were high-pass filtered to remove long-range staining gradients and the contrast was automatically leveled using Adobe Photoshop (registered trademark). An automated grid montage was acquired to obtain TEM statistics in an unbiased manner. Negative staining EM tomography was used as a visualization technique for detailed information on the selected particles. Tilt series were performed from -30° to +30°, and micrographs were acquired at 2° intervals. The tilt series were processed with Etomo (IMOD) to obtain tomograms 30 . The micrographs were aligned with each other by calculating the cross-correlation of consecutive tilt series images. Then, a tomographic image was generated using filtered back-projection. A Gaussian filter was used with a cutoff between 0.25 and 0.5 and a falloff of 0.035.

[0167] Negative staining data processing: The inventors processed the micrographs in CryoSparc 31 and estimated the contrast transfer function (CTF) with CTFFIND4 32 . A combination of manual picking and TOPAZ automatic picking 33 was used to extract particles consisting of different numbers of monomers. The particles were subjected to multiple rounds of 2D classification, sorted, and class average images with increased signal-to-noise ratio were created. The inventors evaluated the distribution of the assemblies through the assignment and manual inspection of particles in specific 2D classes. The inventors measured the dimensions of different types of assemblies based on 2D class average image data using FIJI 34 .

[0168] Preparation of Cryo-Grids and Acquisition of Cryo-EM Images: The inventors vitrified each cryo-EM sample for triangular DNA constructs using a Vitrobot Mark IV (Thermo Scientific). 4 μl of sample was applied to a glow discharge C-Flat grid (Protochips) (Table 6), blotted, and rapidly frozen using the following Vitrobot settings: 22°C temperature, 100% relative humidity, 2–2.5 seconds blotting time, and -1 blotting force. For conical assemblies, a double blotting method was used, consisting of 4 μl sample application, 60-second incubation on the grid, manual blotting, followed by two rounds of sample application, semi-automatic blotting, and rapid freezing. Using a Falcon 3 direct detector (Thermo Scientific) on a Cs-corrected (CEOS) Titan Krios G 2 electron microscope (Thermo Scientific) operating at 300 kV, and EPU software (Thermo Scientific), videos consisting of 10–13 frames were acquired with a cumulative dose of approximately 50 e / m² Å and enlarged pixel sizes of 2.28 Å and 1.79 Å (Table 5). Particle orientation bias was reduced using acquisition with a tilted stage.

[0169] Cryo-EM Data Processing The inventors primarily processed cryo-EM data using the Relion 4 software suite. 35,36 For motion correction of videos and CTF estimation, the inventors have implemented Relion and CTFFIND4, respectively. 32 The inventors used TOPAZ to semi-automatically select particles. 33The particles were extracted, and misselected grid-contaminated and damaged particles were removed via multiple rounds of 2D analysis. Using a low-resolution abu initio initial model created in Relion, structural heterogeneity was addressed via 3D classification, and a 3D purified map was reconstructed. The inventors received the polished and purified set of particles and reconstructed a higher-resolution 3D refined map by applying motion correction and dose weighting for each particle. The inventors post-processed the map by applying a low-resolution mask and Fourier shell correlation (FSC) estimation-based low-pass filtering and sharpening using the 0.143FSC criterion. In Triangle 2 Version 1, the inventors used CryoSparc 31 The final map, including post-processing, was reconstructed using ChimeraX. The dimensions of the 3D electron density map were 3D measured and... 37 The image was rendered using [this method].

[0170] [Table 9] [Table 10]

[0171] References (1) Este, J.; Telenti, A. HIV Entry Inhibitors. The Lancet 2007, 370(9581), 81-88. https: / / doi.org / 10.1016 / S0140-6736(07)61052-6. (2) Pelegrin, M.; Naranjo-Gomez, M.; Piechaczyk, M. Antiviral Monoclonal Antibodies: Can They Be More Than Simple Neutralizing Agents? Trends Microbiol.2015,23(10),653-665.https: / / doi.org / 10.1016 / j.tim.2015.07.005. (3) Tyssen, D.; Henderson, S. A.; Johnson, A.; Sterjovski, J.; Moore, K.; La, J.; Zanin, M.; Sonza, S.; Karellas, P.; Giannis, M. P.; Krippner, G.; Wesselingh, S.; McCarthy, T.; Gorry, P. R.; Ramsland, P. A.; Cone, R.; Paull, J. R. A.; Lewis, G. R.; Tachedjian, G. Structure Activity Relationship of Dendrimer Microbicides with Dual Action Antiviral Activity. PLoS ONE 2010, 5(8), e12309. https: / / doi.org / 10.1371 / journal.pone.0012309. (4) Price, C. F.; Tyssen, D.; Sonza, S.; Davie, A.; Evans, S.; Lewis, G. R.; Xia, S.; Spelman, T.; Hodsman, P.; Moench, T. R.; Humberstone, A.; Paull, J. R. A.; Tachedjian, G. SPL7013 Gel (VivaGel (trademark)) Retains Potent HIV-1 and HSV-2 Inhibitory Activity Following Vaginal Administration in Humans. PLoS ONE 2011, 6(9), e24095. https: / / doi.org / 10.1371 / journal.pone.0024095. (5) Zelikin, A. N.; Stellacci, F. Broad-Spectrum Antiviral Agents Based on Multivalent Inhibitors of Viral Infectivity. Adv. Healthc. Mater. 2021, 10(6), 2001433. https: / / doi.org / 10.1002 / adhm.202001433. (6)Cagno,V.;Andreozzi,P.;DAlicarnasso,M.;Jacob Silva,P.;Mueller,M.;Galloux,M.;Le Goffic,R.;Jones,S.T.;Vallino,M.;Hodek,J.;Weber,J.;Sen,S.;Janecek,E.-R.;Bekdemir,A.;Sanavio,B.;Martinelli,C.;Donalisio,M.;Rameix Welti,M.-A.;Eleouet,J.-F.;Han,Y.;Kaiser,L.;Vukovic,L.;Tapparel,C.;Kral,P.;Krol,S.;Lembo,D.;Stellacci,F.Broad-Spectrum Non-Toxic Antiviral Nanoparticles with a Virucidal Inhibition Mechanism.Nat.Mater.2018,17(2),195-203.https: / / doi.org / 10.1038 / nmat5053. (7)Al-Mahtab,M.;Bazinet,M.;Vaillant,A.Safety and Efficacy of Nucleic Acid Polymers in Monotherapy and Combined with Immunotherapy in Treatment-Naive Bangladeshi Patients with HBeAg+ Chronic Hepatitis B Infection.PLOS ONE 2016,11(6),e0156667.https: / / doi.org / 10.1371 / journal.pone.0156667. (8)Kwon,P.S.;Ren,S.;Kwon,S.-J.;Kizer,M.E.;Kuo,L.;Xie,M.;Zhu,D.;Zhou,F.;Zhang,F.;Kim,D.;Fraser,K.;Kramer,L.D.;Seeman,N.C.;Dordick,J.S.;Linhardt,R.J.;Chao,J.;Wang,X.Designer DNA Architecture Offers Precise and Multivalent Spatial Pattern-Recognition for Viral Sensing and Inhibition.Nat.Chem.2020,12(1),26-35.https: / / doi.org / 10.1038 / s41557-019-0369-8. (9)Ren,S.;Fraser,K.;Kuo,L.;Chauhan,N.;Adrian,A.T.;Zhang,F.;Linhardt,R.J.;Kwon,P.S.;Wang,X.Designer DNA Nanostructures for Viral Inhibition.Nat.Protoc.2022,17(2),282-326.https: / / doi.org / 10.1038 / s41596-021-00641-y. (10)Chauhan,N.;Xiong,Y.;Ren,S.;Dwivedy,A.;Magazine,N.;Zhou,L.;Jin,X.;Zhang,T.;Cunningham,B.T.;Yao,S.;Huang,W.;Wang,X.Net-Shaped DNA Nanostructures Designed for Rapid / Sensitive Detection and Potential Inhibition of the SARS-CoV-2 Virus.J.Am.Chem.Soc.2022,jacs.2c04835.https: / / doi.org / 10.1021 / jacs.2c04835. (11)Sigl,C.;Willner,E.M.;Engelen,W.;Kretzmann,J.A.;Sachenbacher,K.;Liedl,A.;Kolbe,F.;Wilsch,F.;Aghvami,S.A.;Protzer,U.;Hagan,M.F.;Fraden,S.;Dietz,H.Programmable Icosahedral Shell System for Virus Trapping.Nat.Mater.2021,20(9),1281-1289.https: / / doi.org / 10.1038 / s41563-021-01020-4. (12)Monferrer,A.;Kretzmann,J.A.;Sigl,C.;Sapelza,P.;Liedl,A.;Wittmann,B.;Dietz,H.Broad-Spectrum Virus Trapping with Heparan Sulfate-Modified DNA Origami Shells.https: / / doi.org / 10.1021 / acsnano.1c11328. (13)Dadonaite,B.;Vijayakrishnan,S.;Fodor,E.;Bhella,D.;Hutchinson,E.C.Filamentous Influenza Viruses.J.Gen.Virol.2016,97(8),1755-1764.https: / / doi.org / 10.1099 / jgv.0.000535. (14)Wagenbauer,K.F.;Sigl,C.;Dietz,H.Gigadalton-Scale Shape-Programmable DNA Assemblies.Nature 2017,552(7683),78-83.https: / / doi.org / 10.1038 / nature24651. (15)Douglas,S.M.Self-Assembly of DNA into Nanoscale Three-Dimensional Shapes.Nature 459(2009)6. (16)Simmons,C.R.;Zhang,F.;Birktoft,J.J.;Qi,X.;Han,D.;Liu,Y.;Abdallah,H.;Hernandez,C.;Ohayon,Y.;Seeman,N.C.;Yan,H.Construction and Structure Determination of a Three-Dimensional DNA Crystal.J.Am.Chem.Soc.23. (17)Hahn,J.;Wickham,S.F.J.;Shih,W.M.;Perrault,S.D.Addressing the Instability of DNA Nanostructures in Tissue Culture.2014,8(9),11. (18)Laukova,L.;Konecna,B.;Janovicova,L.;Vlkova,B.;Celec,P.Deoxyribonucleases and Their Applications in Biomedicine.Biomolecules 2020,10(7),1036.https: / / doi.org / 10.3390 / biom10071036. (19)Gerling,T.;Kube,M.;Kick,B.;Dietz,H.Sequence-Programmable Covalent Bonding of Designed DNA Assemblies.Sci.Adv.2018,4(8),eaau1157.https: / / doi.org / 10.1126 / sciadv.aau1157. (20)Ponnuswamy,N.;Bastings,M.M.C.;Nathwani,B.;Ryu,J.H.;Chou,L.Y.T.;Vinther,M.;Li,W.A.;Anastassacos,F.M.;Mooney,D.J.;Shih,W.M.Oligolysine-Based Coating Protects DNA Nanostructures from Low-Salt Denaturation and Nuclease Degradation.Nat.Commun.2017,8(1),15654.https: / / doi.org / 10.1038 / ncomms15654. (21)Anastassacos,F.M.;Zhao,Z.;Zeng,Y.;Shih,W.M.Glutaraldehyde Cross-Linking of Oligolysines Coating DNA Origami Greatly Reduces Susceptibility to Nuclease Degradation.J.Am.Chem.Soc.2020,142 (7),3311-3315.https: / / doi.org / 10.1021 / jacs.9b11698. (22)Dreyfuss,J.L.;Regatieri,C.V.;Jarrouge,T.R.;Cavalheiro,R.P.;Sampaio,L.O.;Nader,H.B.Heparan Sulfate Proteoglycans:Structure,Protein Interactions and Cell Signaling.An.Acad.Bras.Cienc.2009,81(3),409-429.https: / / doi.org / 10.1590 / S0001-37652009000300007. (23)Praetorius,F.;Kick,B.;Behler,K.L.;Honemann,M.N.;Weuster-Botz,D.;Dietz,H.BiotechnologicalMass Production of DNA Origami.Nature 2017,552(7683),84-87.https: / / doi.org / 10.1038 / nature24650. (24)Fu,J.;Liu,M.;Liu,Y.;Woodbury,N.W.;Yan,H.Interenzyme Substrate Diffusion for an Enzyme Cascade Organized on Spatially Addressable DNA Nanostructures.J.Am.Chem.Soc.2012,134(12),5516-5519.https: / / doi.org / 10.1021 / ja300897h. (25)Pan,K.;Boulais,E.;Yang,L.;Bathe,M.Structure-Based Model for Light-Harvesting Properties of Nucleic Acid Nanostructures.Nucleic Acids Res.2014,42(4),2159-2170.https: / / doi.org / 10.1093 / nar / gkt1269. (26)Gopinath,A.;Miyazono,E.;Faraon,A.;Rothemund,P.W.K.Engineering and Mapping Nanocavity Emission via Precision Placement of DNA Origami.Nature 2016,535(7612),401-405.https: / / doi.org / 10.1038 / nature18287. (27)Gopinath,A.;Thachuk,C.;Mitskovets,A.;Atwater,HA;Kirkpatrick,D.;Rothemund,PWKAbsolute and Arbitrary Orientation of Single-Molecule Shapes.Science 2021,371(6531),eabd6179.https: / / doi.org / 10.1126 / science.abd6179. (28)Engelhardt,FAS;Praetorius,F.;Wachauf,CH;Brueggenthies,G.;Kohler,F.;Kick,B.;Kadletz,KL;Pham,PN;Behler,KL;Gerling,T.;Dietz,H. Scaffolds.ACS Nano 2019,13(5),5015-5027.https: / / doi.org / 10.1021 / acsnano.9b01025. (29)Dreyfus,J.;Laursen,NS;Quaks,T.;Zuijdgeest,D.;Khayat,R.;Ekiert,DC;Lee,JH;Metlagel,Z.;Bujny,MV;Jongeneelen,M.;van der Vlugt,R.;Lamrani,M.;Korse,HJWM;Geelen,E.;Sahin,Oe.;Siewerts,M.;Brakenhoff,JPJ;Vogels,R .;Li,OTW;Poon,LLM;Peiris,M.;Koudstaal,W.;Ward,AB;Wilson,IA;Goudsmith,J.;Friesen,RHEHighly Conserved Protective Epitopes on Influenza B Viruses.Science 2012,337(6100),1343-1348.https: / / doi.org / 10.1126 / science.1222908. (30)Kremer,J.R.;Mastronarde,D.N.;McIntosh,J.R.Computer Visualization of Three-Dimensional Image Data Using IMOD.J.Struct.Biol.1996,116(1),71-76.https: / / doi.org / 10.1006 / jsbi.1996.0013. (31)Punjani,A.;Rubinstein,J.L.;Fleet,D.J.;Brubaker,M.A.CryoSPARC:Algorithms for Rapid Unsupervised Cryo-EM Structure Determination.Nat.Methods 2017,8. (32)Rohou,A.;Grigorieff,N.CTFFIND4:Fast and Accurate Defocus Estimation from Electron Micrographs.J.Struct.Biol.2015,192(2),216-221.https: / / doi.org / 10.1016 / j.jsb.2015.08.008. (33)Bepler,T.;Morin,A.;Rapp,M.;Brasch,J.;Shapiro,L.;Noble,A.J.;Berger,B.Positive-Unlabeled Convolutional Neural Networks for Particle Picking in Cryo-Electron Micrographs.Nat.Methods 2019,16(11),1153-1160.https: / / doi.org / 10.1038 / s41592-019-0575-8. (34)Schindelin,J.;Arganda-Carreras,I.;Frise,E.;Kaynig,V.;Longair,M.;Pietzsch,T.;Preibisch,S.;Rueden,C.;Saalfeld,S.;Schmid,B.;Tinevez,J.-Y.;White,D.J.;Hartenstein,V.;Eliceiri,K.Tomancak,P.;Cardona,A.Fiji:An Open-Source Platform for Biological-Image Analysis.Nat.Methods 2012,9(7),676-682.https: / / doi.org / 10.1038 / nmeth.2019. (35)Scheres,S.H.W.A Bayesian View on Cryo-EM Structure Determination.J.Mol.Biol.2012,415(2),406-418.https: / / doi.org / 10.1016 / j.jmb.2011.11.010. (36)Kimanius,D.;Dong,L.;Sharov,G.;Nakane,T.;Scheres,S.H.W.New Tools for Automated Cryo-EM Single-Particle Analysis in RELION-4.0.Biochem.J.2021,478(24),4169-4185.https: / / doi.org / 10.1042 / BCJ20210708. (37)Pettersen,E.F.;Goddard,T.D.;Huang,C.C.;Meng,E.C.;Couch,G.S.;Croll,T.I.;Morris,J.H.;Ferrin,T.E.UCSF ChimeraX:Structure Visualization for Researchers,Educators,and Developers.13. (38)Douglas,S.M.;Marblestone,A.H.;Teerapittayanon,S.;Vazquez,A.;Church,G.M.;Shih,W.M.Rapid Prototyping of 3D DNA-Origami Shapes with CaDNAno.Nucleic Acids Res.2009,37(15),5001-5006.https: / / doi.org / 10.1093 / nar / gkp436. (39)Zimmerman,S.B.The Three-Dimensional Structure of DNA.Annu.Rev.Biochem.1982,51(1),395-427. (40)X.C.Bai,T.G.Martin,S.H.Scheres,H.Dietz,Cryo-EM structure of a 3D DNA-origami object.Proceedings of the National Academy of Sciences of the United States of America 109,20012-20017(2012). (41.C.Gortazar et al.,Crossing the interspecies barrier:opening the door to zoonotic pathogens.PLoS pathogens 10,e1004129(2014). (42)S.S.Morse et al.,Prediction and prevention of the next pandemic zoonosis.Lancet 380,1956-1965(2012). (43)J.J.O’Brien,D.M.Campoli-Richards,Acyclovir.An updated review of its antiviral activity,pharmacokinetic properties and therapeutic efficacy.Drugs 37,233-309(1989). (44)J.LaBonte,J.Lebbos,P.Kirkpatrick,Enfuvirtide.Nat Rev Drug Discov 2,345-346(2003). (45)W.L.Davies et al.,Antiviral Activity of 1-Adamantanamine(Amantadine).Science 144,862-863(1964). (46)Y.K.Gupta,M.Meenu,P.Mohan,The Tamiflu fiasco and lessons learnt.Indian J Pharmacol 47,11-16(2015). (47)J.M.Steichen et al.,A generalized HIV vaccine design strategy for priming of broadly neutralizing antibody responses.Science 366,(2019). (48)K.O.Saunders et al.,Targeted selection of HIV-specific antibody mutations by engineering B cell maturation.Science 366,(2019). (49)R.Iinuma et al.,Polyhedra self-assembled from DNA tripods and characterized with 3D DNA-PAINT.Science 344,65-69(2014). (50)P.W.K.Rothemund,Folding DNA to create nanoscale shapes and patterns.Nature 440,297-302(2006). (51)S.M.Douglas et al.,Self-assembly of DNA into nanoscale three-dimensional shapes.Nature 459,414-418(2009). (52)C.E.Castro et al.,A primer to scaffolded DNA origami.Nature methods 8,221-229(2011). (53)R.Veneziano et al.,Designer nanoscale DNA assemblies programmed from the top down.Science 352,1534(2016). (54)E.Benson et al.,DNA rendering of polyhedral meshes at the nanoscale.Nature 523,441-444(2015). (55)K.E.Dunn et al.,Guiding the folding pathway of DNA origami.Nature 525,82-86(2015). (56)J.J.Funke,H.Dietz,Placing molecules with Bohr radius resolution using DNA origami.Nature nanotechnology 11,47-52(2016). (57)R.Jungmann et al.,DNA origami-based nanoribbons:assembly,length distribution,and twist.Nanotechnology 22,275301(2011). (58)W.Liu,H.Zhong,R.Wang,N.C.Seeman,Crystalline two-dimensional DNA-origami arrays.Angewandte Chemie 50,264-267(2011). (59)Y.Suzuki,M.Endo,H.Sugiyama,Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures.Nature communications 6,8052(2015). (60)Y.Ke et al.,DNA brick crystals with prescribed depths.Nature chemistry 6,994-1002(2014). (61)C.Maffeo,J.Yoo,A.Aksimentiev,De novo reconstruction of DNA origami structures through atomistic molecular dynamics simulation.Nucleic acids research 44,3013-3019(2016). (62)T.Gerling,K.F.Wagenbauer,A.M.Neuner,H.Dietz,Dynamic DNA devices and assemblies formed by shape-complementary,non-base pairing 3D components.Science 347,1446-1452(2015). (63)V.Cagno,E.D.Tseligka,S.T.Jones,C.Tapparel,Heparan Sulfate Proteoglycans and Viral Attachment:True Receptors or Adaptation Bias? Viruses 11,(2019)596. (64)Zhang,Q.et al.,Cell Discov.6(2020)1-14. (65)Vaillant,A.,Antiviral Res.133(2016)32-40. (66)Cagno,V.et al.,Antimicrob.Agents Chemother.64(2020)e02001-20. (67)T.Gerling,H.Dietz,Reversible Covalent Stabilization of Stacking Contacts in DNA Assemblies.Angewandte Chemie 58,2680-2684(2019). (68)M.Tan and X.Jiang,Subviral particle as vaccine and vaccine platform.Curr Opin Virol.2014 Jun;6:24-33.

Claims

1. A three-dimensional polynucleotide-based open shell [1] (Figure 26) encloses a cavity [2] and includes an opening [3] for accessing the cavity, A three-dimensional polynucleotide-based open shell comprising an n-pyramid [4] formed by n identical copies of a first type acute isosceles triangular frustum t1 [5], where n is an integer selected from 7, 8, 9, 10, 11, 12, 13, 14, and 15, the base [6] of each frustum pointing to the outside of the open shell, the top [7] pointing to the cavity, two large sides [8, 9] of each frustum comprising a first pattern [10] and a second pattern [11] of one or more protrusions and / or one or more receptors, the first and second patterns being complementary to each other, and a small side [12] comprising a third pattern [13] of one or more protrusions and / or one or more receptors, wherein the first type acute isosceles triangular frustum is a self-assembling DNA-based component.

2. The three-dimensional polynucleotide-based open shell according to claim 1, further comprising n copies of a second type of acute isosceles frustum t2[14], wherein a first side[15] of each frustum points to the outside of the open shell, and the opposite side[16] points to the cavity and / or the opening for accessing the cavity, and one plane[17] of the second type of frustum structure[14] includes a fourth pattern[18] of one or more projections and / or one or more receptacles complementary to the third pattern[13].

3. The three-dimensional polynucleotide-based open shell according to claim 1 or 2, wherein the self-assembling DNA-based components comprise 7,500 to 10,500 base pairs and / or the molecular weight of each self-assembling DNA-based component is 4.5 to 7 MDa.

4. The three-dimensional polynucleotide-based open shell according to any one of claims 1 to 3, wherein the upper surface [7] and / or the opposite side [16], if present, includes 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, wherein the binding molecule is selected from an antibody and its antigen-binding fragment, and a construct comprising at least one sulfonated or sulfated polysaccharide group.

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

7. The three-dimensional polynucleotide-based open shell according to claim 5, wherein 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, wherein the binding molecule is independently selected from the list of heparin, heparan sulfate, hybrid heparan sulfate, carrageenan, cellulose sulfate, dextrin 2-sulfate, aptamer, peptide, host-receptor domain, and sialic acid.

9. A three-dimensional polynucleotide-based open shell according to any one of claims 3 to 8, wherein each truncated pyramidal mass contains 1 to 45 of the aforementioned attachment sites.

10. The three-dimensional polynucleotide-based open shell according to claim 9, wherein each truncated pyramidal mass includes 3 to 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 linked to the binding molecule and complementary to the first single-stranded oligonucleotide.

13. A three-dimensional polynucleotide-based open shell according to any one of claims 1 to 12, wherein each of the first type and, if present, the second type of acute isosceles triangular frustum is a DNA-based nanostructure formed by self-assembling DNA-based components.

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, each of which oligonucleotides is complementary to either a single contiguous DNA sequence stretch or at least two non-contiguous DNA sequence stretches on the single-stranded DNA template.

15. A 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. A three-dimensional polynucleotide-based open shell according to any one of claims 1 to 15, further comprising chemical crosslinking between different truncated pyramidal structures.

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

18. A three-dimensional polynucleotide-based open shell according to any one of claims 1 to 17, further comprising coating 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, further comprising crosslinking of the free amino groups of the polylysine.

22. The crosslinking is by an alkanedialdehyde, as described in claim 21, for a three-dimensional polynucleotide-based open shell.

23. The crosslinking is by glutaraldehyde, as described in claim 22, for a three-dimensional polynucleotide-based open shell.

24. A three-dimensional polynucleotide-based open shell according to any one of claims 1 to 23, for use in the treatment of a patient who is infected, suspected of being infected, or at risk of being infected with a virus, viral particle, or subviral particle.

25. A composition comprising a mixture of 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 a three-dimensional polynucleotide-based open shell having an n value in the range of 9 to 13 and a maximum value in the range of 9 to 11.

27. The composition according to claim 25 or 26 for use in the treatment of a patient who is infected with, suspected of being infected with, or at risk of being infected with, a virus, a viral particle, or a subviral particle.

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

29. A method for treating a patient who is infected with, suspected of being infected with, or at risk of being infected with, a virus, a viral particle, or a subviral particle, comprising the step of 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 of 26.

30. A method for treating a patient who is infected with or suspected to be infected with a virus, viral particle, or subviral particle, the method comprising the step of contacting the patient or the patient's bodily fluids with a three-dimensional polynucleotide-based open shell according to any one of claims 1 to 23, or the composition according to claim 25 of 26.

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

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