DNA ORIGAMI TRAPS FOR LARGE VIRUSES

FIELD OF THE INVENTION

The present invention relates to a three-dimensional polynucleotide-based open shells for encapsulating a virus, a viral particle or a subviral particle, to a composition comprising a mixture of such three-dimensional polynucleotide-based open shells, to a composition comprising a virus, a viral particle or a subviral particle encapsulated by such three-dimensional polynucleotide-based open shells, and to a method for encapsulating a virus, a viral particle or a subviral particle by using such a three- dimensional polynucleotide-based open shells.

BACKGROUND OF THE INVENTION

Viral infections cause millions of deaths per year globally, enormous suffering and morbidity, and impose huge drains on societies and economies in health care costs, lost work time, and other less easily measured burdens such as mental health issues associated with loss of parents, children, and care givers or stigmatization. Climate change and global migration are projected to increase the threat of viral outbreaks because vectors spread to regions that so far were too cold for them to survive. The burden of virus infections will further increase due to habitat encroachment by humans, urbanization and megacities with increasing population density, increasing travel not only locally but also far distance, and numerous other drivers of disease emergence ⁴¹ . Viruses are the pathogen class most likely to adapt to new environmental conditions because of their short generation time and genetic variability allowing rapid evolution ⁴² . For the majority of viral diseases (~70% of current WHO-listed viruses), no effective treatment is available. The few existing antiviral therapies are almost exclusively targeted to a specific virus and do not allow application against a newly emerging pathogen. In addition, antiviral therapy typically faces the challenge that it must be started very soon after infection to be effective, before the viral load gets too high and caused disease symptoms. Emerging virus threats require a rapid response, but broadly applicable ready-to-use antivirals do not exist.

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

Viruses come in many shapes and sizes. Their dimensions range from the 10 to the 1000 nm scale. For example, adeno-associated virus (AAV) is a rather small icosahedral, non-enveloped virus with an approximate and reproducible diameter of 20 nm per particle. Influenza viruses are enveloped and medium-size viruses with dimensions on the 80 to 150 nm scale. Influenza viruses are also pleomorphic, meaning that the particles may adopt a variety of shapes and dimensions including spherical, peanut-shaped or even filamentous. Mimivirus is a representative of a rather large virus with its ~ 700 nm diameter. For all viruses, attachment to the host cell membrane is a prerequisite for cell penetration, infection, and replication.

Preventing viruses from entering cells is increasingly being considered for the development of antiviral treatments. Examples of virus entry inhibitors include peptides, ¹ antibodies, ² dendrimers, ^3-5 nanoparticles and polymers coated with virus- binding moieties. ⁶ ’ ⁷ The majority of these entry inhibitors function on a molecule-to- molecule basis, meaning that one copy of the antiviral agent targets one viral surface protein. More recently, multivalent antiviral concepts have been put forward that display multiple virus-binding molecules in complex geometries intended to match more mesoscale structural aspects of the target pathogen, as exemplified with virus- binding two-dimensional, ^8-10 and three-dimensional DNA architectures. ^{11 12} Multivalent virus-covering nanoarchitectures offer additional options to leverage avidity effects associated with multivalent interactions between antiviral and virus. Multivalent binding leads to exponential amplification of binding strength with valency and can enable achieving virtually irreversible target binding with individually weak and reversible virus binders. Virus surface alterations that reduce the binding strength of individual binders as for example caused by mutational drift may thus be less problematic in the context of the multivalent antiviral relative to a monovalent binder. It is also conceivable that the virus-binding moieties used in the multivalent nanoarchitectures themselves do not necessarily need to have neutralizing activity, since the entry-inhibitory effect will at least in part be accomplished by the virus- surface occluding material of the DNA nanoarchitecture.

It has previously been found that icosahedral DNA origami half-shells ¹¹ can engulf and neutralize viruses up to 85 nm in diameter by mechanically blocking binding interactions with cell surfaces and therefore preventing the infection of host cells. Since there are many larger human viral pathogens of high relevance such as e.g., Influenza, Corona or Herpes viruses, it was sought to expand that approach to also be able to target such pathogens. Influenza viruses are enveloped viruses with dimensions on the 80 to 200 nm scale that occur in a variety of shapes including spherical, peanut-shaped, and filamentous. ¹³ However, the previously developed virus-engulfing shell prototypes were either too restricted in size and shape to accommodate such virus particles or too cumbersome to produce to be of use in a real-world application.

The genomes of viruses frequently present mutations, which may lead to a diminished, or even potentially abolished, success of treatment options, such as vaccinations. Thus, there is a great need for therapeutic interventions that permit the fast adaptation to new emerging developments with respect to, for example, the infectivity of a given virus. None of the approaches mentioned above are modular and flexible enough to enable a fast adaptation of the structures to mutational changes of the viruses.

Thus, while different strategies for the treatment of viral infections have been developed or suggested up to date, there is still a need for the development of a concept of a generic antiviral drug platform for targeting a variety of viral pathogens. In particular, a concept would be desirable that does not rely on prior detailed knowledge about genetics and properties of the target virus. Additionally, it is of particular importance to develop an antiviral drug platform that is amenable for mass production.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide constructs that enable the encapsulation of a virus, a viral particle or a subviral particle. The solution to that problem, i.e., the use of simple macromolecular building blocks, such as DNA-based nanostructures, has not yet been taught or suggested by the prior art.

Therefore, in one aspect, the disclosure provides a three-dimensional polynucleotide-based open shell [1] (Figure 26) encasing a cavity [2] and comprising an opening [3] for accessing said cavity, comprising an n-gonal pyramid [4] formed by n identical copies of a first type of an acute isosceles triangular prismoid t1 [5], wherein n is an integer selected from 7, 8, 9, 10, 11 , 12, 13, 14 and 15, wherein the base plane [6] of each prismoid points to the outside of said open shell, and the upper plane [7] points to said cavity, wherein the two large side planes [8, 9] of each prismoid contain a first pattern [10] and a second pattern [11] of one or more protrusions and/or one or more receptacles, wherein said first and said second patterns are complementary to each other, and wherein the small side plane [12] comprises a third pattern [13] of one or more protrusions and/or one or more receptacles; wherein said first type of an acute isosceles triangular prismoid is a self- assembling DNA-based building block comprising between 7,500 and 10,500 base pairs.

In another aspect, the present invention relates to the three-dimensional polynucleotide-based open shell according to the present invention for use in the treatment of a patient infected by, suspected to be infected by, or bearing the risk of becoming infected by, a virus, a viral particle or a subviral particle.

In another aspect, the present invention relates to a composition comprising a mixture of a three-dimensional polynucleotide-based open shells according to the present invention, wherein said mixture comprises three-dimensional polynucleotide- based open shells having values of n ranging from 7 to 15.

In another aspect, the present invention relates to the composition according to the present invention for use in the treatment of a patient infected by, suspected to be infected by, or bearing the risk of becoming infected by, a virus, a viral particle or a subviral particle.

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 said macromolecule-based nanostructure with a medium comprising, or suspected to comprise, said virus, said viral particle or said subviral particle.

In another aspect, the present invention relates to a method for the treatment of a patient infected by, suspected to be infected by, or bearing the risk of becoming infected by, a virus, a viral particle or a subviral particle, comprising the step of: administering the three-dimensional polynucleotide-based open shell according to the present invention, or the composition according to the present invention to said patient.

In another aspect, the present invention relates to a method for the treatment of a patient infected by, or suspected to be infected by, a virus, a viral particle or a subviral particle, comprising the step of: contacting said patient, or a bodily fluid of said patient, with the three-dimensional polynucleotide-based open shell according to the present invention, or the composition according to the present invention.

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

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

Other features, objects, and advantages of the compositions and methods herein will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the C10 cone DNA origami design (n-gonal pyramid as defined in claim 1.a) with n=10). (A) Left: Schematical model of the C10 conical shell assembly. Cylinders indicate single DNA double helices. Each cone is designed to contain ten isosceles triangular subunits. Right: Schematics of C10 cones covering virus particles. (B) Schematical model of the subunit design, as implemented with multi-layer DNA origami in square-lattice packing. Arrows indicate shape- complementary docking sides located on sides 1 and 2 (S1 and S2). (C) 3D electron density map determined by single particle cryo electron microscopy revealing close agreement between designed and actual overall shape of the wedge subunit (see Fig. 9 for cryo-EM 3D class averages and field of view micrograph)

Figure 2 shows the characterization of cone assembly. (A) Laser-scanned fluorescence image of a 1 % agarose gel on which cone assembly reaction mixtures were electrophoresed, with samples taken at the indicated time points. The wedge subunit concentration was 5 nM, incubation temperature was 40°C, and the solution contained 25 mM MgCl2. M: marker lane. Sc: M13-8064 scaffold as reference. (B) Exemplary negative stain TEM micrograph showing a field of view with cone assembly products. Inset: schematics of typical orientations in which cones adhere on TEM support grid. Scale bar: 100 nm. (C) Two-dimensional TEM class averages of distinct cone assembly species with base-adhered orientations (1 ). Scale bar: 50 nm. (D) Inner diameter measurements of (1 ) 2D class averages for each cone, as well as their frequency of occurrence. (E) Cryo-EM field of view micrograph showing different orientations of cones. Scale bar: 100 nM. (F) Cryo-EM 3D reconstructions of the C9 and C10 cones, with inner diameters and depth measurements.

Figure 3 shows the stabilization of cone assembly for future in vivo applications. (A) Schematic illustration of the stabilization workflow: UV-point welding, oligolysine- PEG coating, and glutaraldehyde cross-linking of coating. (B) Design schematics showing details of the wedge subunit’s strand diagram to indicate the positioning of additional thymidines (yellow dots) for the UV-point welding of t1 subunits. Diagram was prepared using caDNAno vO.2.4. ³⁸ Blue: scaffold strand, grey: staple strands. (C) Laser-scanned fluorescence image of a 1% agarose gel on which cone assembly reaction mixtures were electrophoresed that had been exposed to irradiation with 310 nm light for the indicated times. The gel was run in the 3 mM MgCl2, which are conditions in which non-crosslinked cones immediately disassemble into wedge subunits (see Ctrl or 0 min lane for example). Inset: zoom into the high-molecular weight circular cone assembly products, with each band attributed to a closed cone with the indicated wedge subunit numbers. (D) Exemplary negative stain TEM images taken of non-irradiated (and thus not stabilized) versus irradiated cones in the presence of the indicated MgCl2 concentrations. Scale bar: 100 nm. (E) Exemplary negative stain TEM images taken of solely UV-point welded cone assemblies treated with DNase I (0.001 U/μL) compared to samples that were additionally coated with oligolysine-PEG (1 :0.6, P:N ratio) and chemically cross-linked with glutaraldehyde. Scale bar: 100 nm.

In this context, it should be noted that with respect to Figures 3B, 24 and 25 (see below), those figures show a schematic view of part of the complex arrangements of the different oligonucleotides forming the polynucleotide-based open shells of the present invention. All oligonucleotides used in forming these polynucleotide-based open shells are listed in Tables 1 to 3 and are included in the Sequence Listing. Thus, Tables 1 to 3 contain all sequence information needed in order to generate the nanostructures schematically shown in Figures 3B, 24 and 25, which are included for illustration purposes only. No additional sequence information is included in those figures.

Figure 4 shows the engulfing of Influenza virus particles with cones. (A) Schematics of how cones may be functionalized with virus binding moieties. Red: single stranded DNA extensions called ‘handles’. Blue: DNA-tagged antibodies. (B) Influenza A/PR/8/34 virus trapping with cone assemblies featuring six copies of CR9114 antibodies per wedge subunit. Negative stain TEM images of single virus particles covered with different number of cones. Depending on the size and overall shape of the virus particles, up to three cones coordinated to cover the entirety of spherical/peanut shaped viruses, and even more copies of cones adapted to cover a filamentous Influenza particle. Scale bar: 50 nm. (C) Negative stain TEM images of cones coordinating to trap more than one virus particle at a time. Scale bar: 50 nm. (D) Slices through a single particle 3D tomogram of an Influenza virus fully engulfed by two cones in a sandwich-like assembly, acquired with a negative-staining TEM tilt series. Scale bar: 25 nm.

Figure 5 shows spiked cone assemblies with enhanced surface coverage. (A, B) Schematical model of the spiked cone design that utilizes a second wedge block (t2) designed to assemble onto the cone’s base. (C) Exemplary negative stain TEM micrographs of spiked cone assemblies in different distinct views. (D) Exemplary TEM micrographs showing Influenza A/PR/8/34 virus particles engulfed in spiked cone assemblies functionalized with 6x CR9114 antibodies per wedge subunit. (E) Slices of a negative stain 3D TEM tomogram of a single Influenza virus particle fully engulfed by a single spiked cone, achieving a better surface coverage than non- spiked cones. All scale bars: 50 nm.

Figure 6 shows the schematic representations of design parameters for t1 and t2. (A) Cross-section of 3x6 DNA helices in a square lattice array, in both straight and tilted configurations. (B) Representation of corner angles (a and p) and lengths of the reference helices (a _x and b _x ). (C) Representation of single-stranded DNA loops bridging a corner design. (D) Representation of a beveled angle corner design.

Figure 7 shows the Cryo-EM determination of t1 version 1. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) 3D histograms representing the orientational distribution of particles. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

Figure 8 shows the Cryo-EM determination of t1 version 2. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) 3D histograms representing the orientational distribution of particles. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

Figure 9 shows the Cryo-EM determination of t1 version 3. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) 3D histograms representing the orientational distribution of particles. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

Figure 10 shows Cryo-EM electron density maps of t1 and t2 triangles. Cryo-EM was used to validate the DNA origami designs in an iterative process. It allowed to correct the twist of first versions into nearly twist-free objects (last versions). Figure 11 shows negative stain TEM of t1’s folding reaction crude. This micrograph shows how t1 triangles start to assemble into cones during the folding reaction. Extra staples from the folding can be seen in the background. Scale bar: 100 nm.

Figure 12 shows negative stain TEM of unspecific stacking of cones induced by high ionic strength. Lateral and top views of unspecific cone stacking. Scale bar: 100 nm.

Figure 13 shows 2D class averages of cones extracted from negative stain TEM. Vertex-adhered cones have larger diameters and frayed circumference compared to base-adhered cones containing the same number of wedge building blocks. Scale bar: 100nm.

Figure 14 shows Cryo-EM of cones. (A) Different views of the electron density map of the C9 cone. Scale bar: 50 nm. (B) Different views of the electron density map of the C10 cone. Scale bar: 50 nm. (C) 3D histograms representing the orientational distribution of C9 cones. (D) Like in C but for C10 cones.

Figure 15 shows 3D measurement of dimensions of cryo-EM reconstructions. (A) C9 cone. (B) C10 cone.

Figure 16 shows a Multibody Analysis of the C9 object. (A) Nine masks (colored, semi-transparent) enclosing the reconstruction of the C9 object used for Multibody Refinement. (B) Principal Component Analysis of refined orientations of individual rigid bodies from a 9-body Multibody Refinement. (C) Distribution of particle weights along the 1st principal component (PC). (D) Reconstructions of two subsets of the particle ensemble. Subset 1 (orange) contains particles with weight value -999 to 0 along PC1 , subset 2 (blue) contains particles with values 0 to 999.

Figure 17 shows negative stain TEM of a negative control for Influenza A/PR/8/34 trapping with cones. Field of view demonstrating no binding of Influenza virus particles without the antibody coating. Scale bar: 100 nm. Figure 18 shows the Cryo-EM determination of t2 version 1 . (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) Histogram representing the orientational distribution of particles. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

Figure 19 shows the Cryo-EM determination of t2 version 2. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) 3D histograms representing the orientational distribution of particles. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

Figure 20 shows the Cryo-EM determination of t2 version 3. (A) Exemplary micrograph. Scale bar: 100 nm. (B) Representative 2D class averages. (C) 3D histograms representing the orientational distribution of particles. (D) FSC plot. (E) Six different views of the electron density map. Scale bar: 25 nm.

Figure 21 shows a cylindrical representation of triangles 1 and 2 assembly features. (A) t1’s side 3 can be functionalized with a protrusion orthogonal to sides 1 and 2 for the assembly of t2, which has a complementary feature in the form of a recess. (B) Dimer representation in two different views.

Figure 22 shows t1-t2 dimer assembly characterization. (A) Exemplary laser- scanned fluorescent image of a 1 .5% agarose gel showing the assembly of t1 with t2 in a 1 :1 ratio over the course of 2 days, with a triangle monomer concentration of 5 nM incubated at 40°C in presence of 25 mM MgCl2. Sc: M13-8064 scaffold as reference. Sides 1 and 2 of t1 were passivated to avoid the cone assembly. (B) % of completely assembled dimers at different time points and different MgCl2 concentrations. The % were extracted from agarose gels like the one shown in (A). Error bars show standard deviations of triplicates.

Figure 23 shows broadband virus trapping with heparan sulfate-mod if ied spiked cones. (A) Schematics of how cones may be functionalized with virus binding moieties. Red: single stranded DNA extensions called ‘handles. Orange: HS polymers. Trapping was performed with spiked cones featuring 12 heparan sulfate moieties per wedge subunit. (B) Exemplary negative stain TEM micrographs showing trapped SARS-CoV-2 and Zika virus-like particles (VLPs). (C) Negative stain TEM micrograph showing trapped Chikungunya VLPs. Due to the smaller size of the CHIK-VLPs, up to three virus particles fit into the large cavity of the spiked cone, which significantly deformed themselves to maximize their contact with the viruses. All scale bars: 50 nm.

Figure 24 shows the caDNAno design diagram for triangle 1 (A) version 1 , (B) version 2, and (C) version 3. Blue: scaffold strand, colorful: staple strands. Designs prepared with caDNAno vO.2.4.

Figure 25 shows the caDNAno design diagram for triangle 2 (A) version 1 , (B) version 2, and (C) version 3. Blue: scaffold strand, colorful: staple strands. Designs prepared with caDNAno vO.2.4.

Figure 26 shows the schematic representation of the three-dimensional polynucleotide-based open shells of the present invention including the reference numbers used in the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides constructs that enable the encapsulation of a virus, a viral particle or a subviral particle.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains.

The terms “comprising” and “including” are used herein in their open-ended and non-limiting sense unless otherwise noted. With respect to such latter embodiments, the term “comprising” thus includes the narrower term “consisting of”. The terms “a” and “an” and “the” and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. Where the plural form is used for compounds, salts, and the like, this is taken to mean also a single compound, salt, or the like.

Therefore, in one aspect, the disclosure provides a three-dimensional polynucleotide-based open shell [1] (the reference numbers refer to Figure 26) encasing a cavity [2] and comprising an n-gonal pyramid [4] formed by n identical copies of a first type of an acute isosceles triangular prismoid t1 [5], wherein n is an integer selected from 7, 8, 9, 10, 11 , 12, 13, 14 and 15, wherein the base plane [6] of each prismoid points to the outside of said open shell, and the upper plane [7] points to said cavity, wherein the two large side planes [8, 9] of each prismoid contain a first pattern [10] and a second pattern [11] of one or more protrusions and/or one or more receptacles, wherein said first and said second patterns are complementary to each other, and wherein the small side plane [12] comprises a third pattern [13] of one or more protrusions and/or one or more receptacles; wherein said first type of an acute isosceles triangular prismoid is a self-assembling DNA-based building block

In a particular embodiment, the self-assembling DNA-based building block comprises between 7,500 and 10,500 base pairs.

In a particular embodiment, the molecular weight of each self-assembling DNA- based building block is between 4.5 and 7 MDa.

In a particular embodiment, the disclosure provides a three-dimensional polynucleotide-based open shell, which is DNA-based.

In the context of the present disclosure, the term “polynucleotide-based open shell, which is DNA-based” refers to a DNA-based nanostructure that is formed by a set of DNA-based macromolecules. DNA-based nanostructures similar to the ones used in accordance with the present invention are described in detail in of WO 2021/165528 and in Sigi et al., loc. cit..

In the context of the present disclosure, the term “DNA” refers to deoxyribonucleic acid composed of a single-strand of monomeric units called nucleotides, wherein each nucleotide is composed of a nitrogen-containing nucleobase, a 2-deoxyribose sugar moiety, and a phosphate group, wherein the individual nucleotides are linked in the single-strand by a phosphate group linking the OH group in position 5’ of a 2-deoxyribose sugar moiety to the OH group in 3’ of a neighboring 2-deoxyribose sugar moiety. In particular embodiments, the nitrogen- containing nucleobases are independently selected from cytosine [C], guanine [G], adenine [A] and thymine [T], In particular embodiments, one or more of the nucleobases are non-canonical bases, in particular a non-canonical base selected from the list of: a modified adenosine, in particular N6-carbamoyl-methyladenine or N6-methyadenine; a modified guanine, in particular 7-deazaguanine or 7- methylguanine; a modified cytosine, N4-methylcytosine, 5-carboxylcytosine, 5- formylcytosine, 5-glycosylhydroxymethylcytosine, 5-hydroxycytosine, or 5- methylcytosine; a modified thymidine, in particular a-glutamyl thymidine or a- putrescinyl thymine; a uracil or a modification thereof, in particular uracil, base J, 5- dihydroxypentauracil; or 5-hydroxymethyldeoxyuracil; deoxyarchaeosine and 2,6- diam inopurine. A stretch of a single-strand of DNA may interact with a complementary stretch of DNA by interaction of complementary nucleobases, wherein cytosine and guanine, and adenine and thymine, are complementary to each other, respectively by forming two (A/T) and three (G/C) hydrogen bonds between the nucleobases. Two single-strands of DNA may be fully complementary to each other, as in the case of genomic DNA, or may be partially complementary to each other, 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- stranded DNA double helix. As is well known, DNA has evolved in nature as carrier of the genetic information encoding proteins. DNA further includes non-coding regions that include regions having regulatory functions. Thus, any DNA-based application usually critically depends on the specific DNA sequence and is almost always only enabled by naming the specific DNA sequence. In contrast, in the context of the present invention, such coding and/or regulatory functions do not play any role and may or may not be present, since the underlying DNA sequences are solely designed and selected in a way that the desired arrangement of double-helical subunits is formed. Thus, in one embodiment any form of a long single-stranded DNA sequence, whether naturally occurring DNA (such as the DNA of a bacteriophage) or synthetically produced DNA may be selected as template, and a set of short single-stranded DNA sequences may be designed, wherein each sequence is complementary to one or more different parts of the template and thus forms one or more double-helical sections. Collectively, all such double-helical sections created by interaction of the full set of short single-stranded DNA sequences with the template, then form the desired three-dimensional arrangement. Starting from a given single-stranded template sequence, the design of a set of complementary can be set up using known techniques, such as, for example, the methods described for the synthesis of megadalton-scale discrete objects with structurally well-defined 3D shapes ¹⁵ ’ ⁴⁰ ’ ⁴⁹ ’ ⁶⁰ . In particular, iterative design with caDNAno ³⁸ paired with elastic-network-guided molecular dynamics simulations ⁶¹ can be used.

In addition to the interaction of complementary nucleobases of different stretches of single-stranded DNA via hydrogen bonds, additional interactions between different DNA strands are possible, including the interactions between the ends of two double- stranded DNA helices by protrusion and recess features using either blunt ends or sticky ends for increased stability and specificity ⁶² , thus enabling the design and the formation of complex DNA-based nanostructures via the shape-complementarity of double-helical subunits. Thus, two three-dimensional arrangements formed in accordance with the previous paragraph, may interact with each other by interactions between double-helical subunits present on the two three-dimensional arrangements, including specific interactions between two three-dimensional arrangements having complementary protrusions and recessions (or knobs and holes).

In a particular embodiment, the DNA-based nanostructure is formed by self- assembling DNA-based building blocks.

In a particular embodiment, each of said self-assembling DNA-based building blocks is formed by a single-stranded DNA template strand and a set of oligonucleotides complementary to said single-stranded DNA template, wherein each of said oligonucleotides is either complementary to one contiguous DNA sequence stretch or to at least two non-contiguous DNA sequence stretches on said single- stranded DNA template.

In a particular embodiment, the DNA-based nanostructure consists of between 4 and 180 of such self-assembling DNA-based building blocks.

In particular embodiments, said single-stranded DNA template is single-stranded DNA of filamentous bacteriophage, or is derived from single-stranded DNA of filamentous bacteriophage.

In the context of the present invention, the term “filamentous bacteriophage” refers to a type of bacteriophage, or virus of bacteria, which is characterized by its filament-like shape that usually contains a genome of circular single-stranded DNA and infects Gram-negative bacteria. Filamentous phage includes Ff phage, such as M13, f1 and fd1 phage, and Pf1 phage.

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

In particular embodiments, said single-stranded DNA is circular.

In the context of the present invention, a single-stranded DNA template that is ’’derived from single-stranded DNA of filamentous bacteriophage” refers to a DNA construct that is derived from a naturally occurring of published DNA sequence of a filamentous bacteriophage by one or more of: (i) opening of the circular structure to 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. While any such variation might have detrimental, or at least rather unpredictable, effects on bacteriophage biology, its infectivity and its ability to propagate, such effects do not play any role in the context of the present invention, since, as already mentioned above, said single-stranded DNA template is only used as naked template without any requirement for having any functional property, and all structural aspects, such as the correct formation the three-dimensional shape of said self-assembling DNA- based building blocks, are implemented by the proper choice of said set of complementary oligonucleotides.

In particular embodiments, said single-stranded DNA template has at least 80 %, particularly at least 90 %, more particularly at least 95 %, sequence identity to the sequence of a naturally occurring or published sequence of a filamentous bacteriophage, in particular to a M13, f1 or fdl phage, in particular to a sequence selected from SEQ ID NO: 1 (M13 8064) and M13 7249 (see SEQ ID NO: 2 of WO 2021/165528). In this context, it should be mentioned that the single-stranded DNA template is used in the present invention as template only, so that the exact sequence does not have any biological role and/or function. Instead, any sequence of similar length could be used, since the setup of the three-dimensional structure of the polynucleotide-based open shell is essentially achieved by synthesizing a set of oligonucleotides having complementarity with two or more sequence stretches on said single-stranded DNA template. That set of complementary oligonucleotides can be designed manually, but is easier by using computer programs such as caDNAno ³⁷ Thus, bacteriophage sequences listed above are given as examples only.

In the context of the present invention, the term “acute isosceles triangular prismoid” refers to a polyhedron, wherein all vertices lie in two parallel planes, which is a triangular prismoid having two planes in the form of acute isosceles triangles. In a particular embodiment, the present invention relates to a DNA-based nanostructure, wherein each said triangular prismoid, is formed by m triangular planes, wherein m is an integer independently selected from 4, 5, 6, 7 and 8, in particular independently selected from 5, 6 and 7, more particularly wherein said integer is 6, wherein the three, or four, respectively, edges of each of said m planes are formed by n parallel stretches of DNA double helices, wherein n is an integer independently selected from 1 , 2, 3, 4, 5 and 6 in particular independently selected from 2, 3, 4 and 5, more particularly independently selected from 3 and 4, wherein each plane is connected to a plane above and/or a plane beyond said plane

(i) by stacking interactions between the DNA double helices forming said planes, and

(ii) partially by DNA stretches within said single-stranded DNA template and/or said oligonucleotides forming said DNA-based building block bridging at least two of said planes, and wherein at least two of the three, or four, respectively, side trapezoids comprise a specific pattern of recesses and/or extrusions formed by missing or additional DNA double helical stretches for specific interaction with a complementary pattern on the side trapezoid of another one of said self-assembling DNA-based building blocks.

In a particular embodiment, the average length of each of the n stretches of DNA double helices in the m planes of a triangular, or rectangular, respectively, prismoid is between 80 and 200 base pairs.

In particular embodiments, said triangular prismoid is a triangular frustum.

In the context of the present 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 has been removed resulting in a plane on the top parallel to the basis of the pyramid.

In a particular embodiment, for at least part of said self-assembling DNA-based building blocks the length of at least one edge of each of said m planes is decreasing from the first to the m ^th plane, so that a bevel angle 0 results between planes perpendicular to said first plane and the trapezoid plane formed by said m edges (see Fig. 6). In particular embodiments, all three trapezoid planes exhibit a bevel angle.

In a particular embodiment, a bevel angle is between 16° and 26°, particularly between 18° and 24°, more particularly between 20° and 22°, most particularly about 20.9°.

In a particular embodiment, said DNA-based nanostructure comprises at least one set of self-assembling DNA-based building blocks, wherein all three, or four, respectively, side trapezoids comprise a specific pattern of recesses and/or extrusions formed by missing or additional DNA double helical stretches for specific interaction with a complementary pattern on the side trapezoid of another one of said self-assembling DNA-based building blocks.

In a particular embodiment, said three-dimensional polynucleotide-based open shell further comprises n copies of a second type of an acute isosceles triangular prismoid t2 [14], wherein a first side [15] of each prismoid points to the outside of said open shell, and the opposite side [16] points to said cavity and/or to said opening for accessing said cavity, wherein one plane [17] of said second type of prismoid structure [14] comprises a fourth pattern [18] of one or more protrusions and/or one or more receptacles which is complementary to said third pattern [13],

In a particular embodiment, said DNA-based nanostructure comprises two sets of self-assembling DNA-based building blocks, in particular the self-assembling DNA- based building blocks t1 and t2.

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

In the context of the present disclosure, the term “RNA” refers to ribonucleic acid composed of a single-strand of monomeric units called nucleotides, wherein each nucleotide is composed of a nitrogen-containing nucleobase, a ribose sugar moiety, and a phosphate group, wherein the individual nucleotides are linked in the single- strand by a phosphate group linking the OH group in position 5’ of a ribose sugar moiety to the OH group in 3’ of a neighboring ribose sugar moiety. In particular embodiments, the nitrogen-containing nucleobases are independently selected from cytosine [C], guanine [G], adenine [A] and uracil [II]. In particular embodiments, one or more of the nucleobases are non-canonical bases, in particular a non-canonical base selected from the list of: pseudouridine, ribothymidine, and inosine. Unlike DNA, RNA is most often in a single-stranded form, but the formation of double-stranded forms is possible by interaction of complementary nucleobases, wherein cytosine and guanine, and adenine and uracil, are complementary to each other, respectively by forming two (A/U) and three (G/C) hydrogen bonds between the nucleobases. In a particular embodiment, the disclosure provides a macromolecule-based nanostructure, which is an RNA-based nanostructure.

In the context of the present invention, the term “cavity” relates to the space enclosed by said DNA-based nanostructure. In particular embodiments, said cavity resembles a sphere, where a spherical segment has been cut off, with the cutting plane being formed by the self-assembling DNA-based building blocks at the borders of said DNA-based nanostructure. In particular embodiments, the cutting plane is a great circle so that the DNA-based nanostructure is a half-shell.

In a particular embodiment, said upper plane [7] and/or, when present, said opposite side [16] comprise one or more attachment sites for the attachment of one or more binding molecules, which are specifically or non-specifically interacting with a virus, a viral particle or a subviral particle.

In particular embodiments, said one or more binding molecules are specifically interacting with said virus, said viral particle or said subviral particle by being able to bind and to inactivate, said viral particle or said subviral particle.

In a particular embodiment, said binding molecules are specifically interacting with a virus, a viral particle or a subviral particle. In particular, said binding molecules are selected from antibodies and antigen-binding fragments thereof comprising at least an antigen-binding site of an antibody, in particular at least a VH domain of an antibody, or at least a combination of a VH and a VL domain of an antibody particularly scFv fragments.

In a particular other embodiment, said binding molecules are non-specifically interacting with a virus, a viral particle or a subviral particle, in particular constructs comprising at least one sulfonated or sulfated polysaccharide group, particularly a construct comprising one or two sulfonated or sulfated polysaccharide groups, more particularly wherein said sulfonated or sulfated polysaccharide is independently selected from the list of heparin, heparan sulfate, hybrid heparan sulfates, carrageenans, cellulose sulfate, dextrin 2-sulfate, aptamers, peptides, host-receptor domains, sialic acid.

In the context of the present application, the term “viral particle” relates to a virus- like particle that resembles the three-dimensional structure of an intact virus without being biologically active, and the term “subviral particle” relates to a smaller virus-like particle smaller particles with less or smaller subunits, which can be produced for some viruses by expressing not all and/or only portions of one or more major viral capsid proteins. These artificial viral particles or subviral particles retain the structures and antigenic properties of their native viruses, including the virus-specific molecular patterns and high density of B-cell and T-cell epitopes to induce potent innate, humoral, and cellular immune responses, respectively, in animals and humans ⁶⁸ .

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

In the context of the present application, the term “sulfonated or sulfated polysaccharide group” relates to a group comprising a polysaccharide comprising at least one sulfated hydroxy group or at least one sulfonated glycosylamino group. Importantly, in addition to targeting specific receptors, many viruses also weakly interact with different biological substances, including sulfated of sulfonated polysaccharides ( ⁶³ ; see Table 4).

In particular embodiments, said polysaccharide comprising at least one sulfated hydroxy group or at least one sulfonated glycosylamino group is independently selected from the list of heparin, heparan sulfate, hybrid heparan sulfates, carrageenans, cellulose sulfate, and dextrin 2-sulfate.

In particular embodiments, said polysaccharide comprising at least one sulfated hydroxy group or at least one sulfonated glycosylamino group consists of between 3 and 15 disaccharide units, in particular 4, 5, 6, 7, 8 of 9 units, particularly 4 or 9 monosaccharide units.

In particular embodiments, said disaccharide units comprise two or three 0- and/or N-sulfonate groups per disaccharide unit, in particular three 0- and/or N- sulfonate groups.

In particular embodiments, said polysaccharide comprising at least one sulfated hydroxy group or at least one sulfonated glycosylamino group is independently selected from heparin, heparan sulfate, and hybrid heparan sulfates.

In the context of the present invention, the terms “heparin” and “heparan sulfate” both relate to a family of linear sulfated, heterogeneous polysaccharides found on the cell membrane and in the extracellular matrix as part of heparan sulfate proteoglycans (HSPGs). They are composed of repeating 1 —> 4 linked disaccharide units, in which one monosaccharide is an a-D-glucosamine residue and the other an uronic acid (or, in a salt form, an uronate). Heparin is a structurally similar polysaccharide found within mast cells as a component of serglycin proteoglycans. Heparan sulfate and heparin can be defined as follows: first, in heparin, the uronates are predominantly a-L-iduronate, whereas in heparan sulfate, the uronates are mainly, [3-D-glucuronates, the C-5 epimers of a-L-iduronate. Second, in heparan sulfate, the D-glucosamine residues are predominantly N-acetylated, whereas in heparin, they are N-sulfonated. Finally, whereas at least 70-80 % of heparin is composed of the disaccharide L-iduronate 2-O-sulfate a(1 —> 4) D-glucosamine No- sulfate, in heparan sulfate around 40-60 % of the disaccharides consist of (1 — >4) D- glucuronate [3 (1 — ► 4) D-glucosamine, that can be either N-acetylated or N- sulfonated. Together, these structural characteristics make heparin more sulfated and, hence, more charged than heparan sulfate. It has become apparent, however, that the designations heparin or heparan sulfate are less clear-cut than this description implies, and that polysaccharides isolated from some 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 structures being a mixture of the “typical” heparin structural elements (L-iduronates; high degree of sulfonation) and the “typical” heparan sulfate structural elements (D-glucuronate; N-acetylation and 6-O-sulfonation).

Heparan sulfate proteoglycans (HSPG) ^{63; 64} are commonly found on the surface of mammalian cells. The weak interactions of viruses with HSPG are conserved across virus families and thus appear generically beneficial for the virus lifecycle. For example, HSPG-virus interactions may enable an infection-enhancing diffusive search of virus particles for their specific host cell receptors on the surface of cells. The interactions of heparan sulfate (HS) with viruses have already been exploited for medical purposes, for example in virus-sequestering coatings of condoms that are based on HS-decorated dendrimers ^3-5 . Other investigations have frequently involved the surface functionalization of nanoparticles and polymers with HS derivatives to create virus-binding complexes with antiviral activity ⁶ ; ^{7; 65; 66} . Commonly, a high level of multivalency is required to increase the strength of binding between the HS- nanoparticles and viruses. The reversible nature of the binding can lead to undesirable unbinding and release of infectious viruses from the virus-sequestering coatings, or the requirement for high concentrations of the therapeutically active agent to be maintained ⁵ .

In particular embodiments, said macromolecule-based nanostructure comprises, on average, between one and 10 binding molecules attached to the interior site of the cavity formed by said macromolecule-based nanostructure, in particular between 4 and 10, in particular four, five, six, seven, eight, nine or ten binding molecules.

In particular embodiments, one or more of said self-assembling DNA-based building blocks is linked to a construct comprising at least one sulfonated or sulfated polysaccharide group pointing to the interior of said cavity, particularly a construct comprising one or two sulfonated or sulfated polysaccharide groups.

In particular embodiments, said three-dimensional polynucleotide-based open shell is a DNA-based nanostructure in accordance with the present invention, wherein said at least one binding molecule is linked to one of said triangular prismoids forming the DNA-based nanostructure in a way that said at least one binding molecule is located on the inside of said DNA-based nanostructure and is pointing into the cavity formed by said DNA-based nanostructure.

In a particular embodiment, each prismoid comprises between 1 and 45, in particular between 1 and 32 of said attachment sites, particular between 3 and 10 attachment sites. In particular embodiments, all prismoids comprise said attachments sites. In other embodiments, only the t1 prismoids comprise said attachments sites, or only the t2 prismoids comprise said attachments sites.

In a particular embodiment, said attachment sites are first single-stranded oligonucleotides.

In a particular embodiment, said binding molecules are attached to said attachment sites by second single-stranded oligonucleotides, which are linked to one or more binding molecules and are complementary to, or otherwise able to enter site- specific interactions with, said first single-stranded oligonucleotides. In particular embodiments, each of said single-stranded oligonucleotides is linked to one binding molecule. In other embodiments, each of said single-stranded oligonucleotides is linked to two binding molecules.

In a particular embodiment, each of said first and of said optional second types of said acute isosceles triangular prismoids is a DNA-based nanostructure formed by self-assembling DNA-based building blocks, in particular wherein said DNA-based nanostructure is formed by a single-stranded DNA template strand and a set of oligonucleotides complementary to said single-stranded DNA template, wherein each of said oligonucleotides is either complementary to one contiguous DNA sequence stretch or to at least two non-contiguous DNA sequence stretches on said single- stranded DNA template.

In particular embodiments, the apex angle of the acute isosceles triangles forming the opposing planes of said acute isosceles triangular prismoids is between 15° and 60°, in particular between 20° to 30°.

In a particular embodiment, n is an integer selected from 9, 10, 11 , 12 and 13.

In a particular embodiment, said three-dimensional polynucleotide-based open shell further comprises chemical crosslinks between different prismoids further comprises one or more cross-linkages within one of said triangular prismoids, and/or between two of said triangular prismoids.

In the context of the present invention, the term “cross-linkage” refers to any permanent or intermittent linkage within one of said triangular prismoids, and/or between two of said triangular prismoids. Any such linkage may be achieved a priori by linking two of the oligonucleotides being used for forming the self-assembling DNA-based building blocks prior to the assembly, or a posteriori, e. g. by chemically or photochemically adding linkages between different parts of the three-dimensional nanostructure. Permanent linkages may, for example, be created by photochemically cross-linking T residues appropriately positioned in the structure under formation of covalent cyclobutane pyrimidine dimer (CPD) bonds ¹⁹ , and intermittent linkages may, for example, be created by photochemically cross-linking the blunt ends of two double-helical subunits between a 3-cyanovinylcarbazole (cnvK) moiety positioned at a first blunt end and a thymine residue (T) positioned at the other blunt end ⁶⁷ .

In a particular embodiment, said three-dimensional polynucleotide-based open shell further comprises chemical crosslinks between different triangular prismoids. In a particular embodiment, said chemical crosslinks are obtained by UV irradiation.

In a particular embodiment, said three-dimensional polynucleotide-based open shell further comprises a coating of the outer surface of said open shell with a polycationic molecule.

In a particular embodiment, said polycationic molecule is a polylysine, particularly polylysine-PEG.

In a particular embodiment, said three-dimensional polynucleotide-based open shell further comprises cross-links of free amino groups of said polylysine, particularly with an alkane dialdehyde, in particular with glutaraldehyde.

In a particular embodiment, said opening [3] has a diameter [19] between 100 and 200 nm.

In the context of the present invention, the term “diameter” refers to the diameter [19] as shown in Figure 26.

In particular embodiments, three-dimensional polynucleotide-based open shell has a molecular weight between 30 MDa and 80 MDa (t1 only), particularly between 40 MDa and 70 MDa, and between 60 MDa and 160 MDa (t1 plus t2), particularly between 80 MDa and 140 MDa.

In particular embodiments, the volume of the cavity encased by said three- dimensional polynucleotide-based open shell (in nm ³ ) is between 80,000 and 200,000, particularly between 100,000 and 140,000.

In another aspect, the present invention relates to a composition comprising a mixture of a three-dimensional polynucleotide-based open shells according to the present invention, wherein said mixture comprises three-dimensional polynucleotide- based open shells having values of n ranging from 7 to 15. particularly ranging from 9 to 13, with a maximum in the range of 9 to 11 . In another aspect, the present invention relates to the composition according to the present invention for use in the treatment of a patient infected by, suspected to be infected by, or bearing the risk of becoming infected by, a virus, a viral particle or a subviral particle.

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 said macromolecule-based nanostructure with a medium comprising, or suspected to comprise, said virus, said viral particle or said subviral particle.

In particular embodiments, said method is for removing said virus, said viral particle or said subviral particle from said medium. In particular embodiment, said method is for encapsulating said virus, said viral particle or said subviral particle in order to transport said virus, said viral particle or said subviral particle.

In particular embodiments, said method for removing said virus, said viral particle or said subviral particle relates to a method for the treatment of a patient infected by, suspected to be infected by, or bearing the risk of becoming infected by, said virus, said viral particle or said subviral particle, comprising the step of: administering the three-dimensional polynucleotide-based open shell according to the present invention, or the composition according to the present invention to said patient.

In particular embodiments, said method for the treatment of a patient infected by, or suspected to be infected by, a virus, a viral particle or a subviral particle, comprises the step of: contacting said patient, or a bodily fluid of said patient, with the three-dimensional polynucleotide-based open shell according to the present invention, or the composition according to the present invention.

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

In particular embodiments, said composition is formed in a process of removing said virus, said viral particle or said subviral particle from a medium containing said virus, said viral particle or said subviral particle. In particular other embodiments, said composition is formed in a process of incorporating said virus, said viral particle or said subviral particle as cargo in said three-dimensional polynucleotide-based open shell.

In another aspect, the disclosure provides a composition comprising a cargo different from a virus, a viral particle or a subviral particle, where said cargo, such as a complex macromolecule, is encapsulated by a three-dimensional polynucleotide- based open shell according to the present invention. In particular embodiments, said cargo is a cytokine. In particular embodiments, said cytokine is interleukin-6.

In yet another aspect, the disclosure provides a method for encapsulating a cargo different from a virus, a viral particle or a subviral particle, such as a complex macromolecule, comprising the steps of: providing a three-dimensional polynucleotide-based open shell according to the present invention, and contacting said three-dimensional polynucleotide-based open shell with a medium comprising, or suspected to comprise, said cargo. In particular embodiments, said cargo is a cytokine. In particular embodiments, said cytokine is interleukin-6.

TABLES 1 to 3: Sequence listing.

TABLE 1 : Temp ate Sequence

TABLE 2: Staple sequences for triangle 1

TABLE 2B: Staple sequences for triangle 1, version 2

TABLE 3: Staple sequences for triangle 2

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

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

The following Examples illustrates the invention described above, but is not, however, intended to limit the scope of the invention in any way. Other test models known as such to the person skilled in the pertinent art can also determine the beneficial effects of the claimed invention.

EXAMPLES

Introduction

Virus-enveloping macromolecular shells or tilings can in principle prevent viruses from entering cells. Here we describe the design and assembly of a cone-shaped DNA origami higher-order assembly that can engulf and tile the surface of pleomorphic virus samples larger than 100 nm. We determine the structures of subunits and of complete cone assemblies using cryo-EM; and establish stabilization treatments to enable usage in in vivo conditions. We use the cones exemplarily to engulf Influenza A virus particles, and SARS-CoV-2, Chikungunya and Zika virus-like particles. Depending on the relative dimensions of cone to virus particles, multiple virus particles may be trapped per single cone, and multiple cones can also tile and adapt to the surface of aspherical virus particles. The cone assemblies form with high yields, require little purification, and are amenable for mass production, which is a key requirement for future real-world uses including as an antiviral agent.

To overcome the limitations referred to in the section describing the background of the invention, we here describe an efficiently assembling DNA origami based macromolecular shell system that can engulf pleomorphic viral pathogens larger than 100 nm in diameter as exemplified by Influenza A viruses. Our design concept considers the self-limiting oligomerization of wedge-shaped building blocks into cones. This expansion of a previous implementation of planar finite size assemblies ¹⁴ uses a minimized number of subunit types which reduces the complexity of the assembly process. The resulting high yields of assembly make the cone system amenable to mass production as needed for future real-world uses as an antiviral.

RESULTS AND DISCUSSION

Our cone assemblies are designed to form from multiple copies of a wedge-shaped building block (t1 ) (see supporting information for design details). The wedge building block can oligomerize via two distinct self-complementary edges at opposite faces. Oligomerization of the wedges leads to circular assemblies that close upon themselves. Given the designed geometry of the wedge, we expect the cone to have ten facets (Fig. 1 A, B). The diameter of the base of the cone made of the ten wedges was designed to measure ~120 nm, so that two copies of a cone would, for example, be sufficiently large to enclose an Influenza virus particle (~ 80-200 nm) in a sandwich- like assembly (Fig. 1A, right). We implemented the wedge building block with multi-layer DNA origami in square- lattice helical packing. ^{15 16} We assembled the objects using the methods of DNA origami and used single-particle cryogenic electron microscopy (cryo-EM) to improve and validate the design of our wedge subunit in an iterative process (Fig. 7-9). The 3D electron density maps we determined for the single wedge particles revealed the designed overall shape of the triangular building block and the shape-complementary docking features (Fig. 1 C). Our initial wedge design displayed a pronounced global twist deformation, which we then corrected to give a nearly twist-free shape (Fig. 10).

We triggered the oligomerization of wedge subunits into cones by increasing the ionic strength of the solution after folding of the wedge building blocks from its constituent DNA staple and scaffold strands. Oligomerization can also occur concomitantly during the wedge assembly reaction, depending on the ionic condition used (Fig. 11 ). We monitored the formation of cones in a time-dependent fashion by gel-electrophoretic mobility analysis (Fig. 2A), where the appearance and disappearance of bands towards increasingly lower electrophoretic mobilities reflected the progressive oligomerization of the wedge subunits as a function of incubation time. Eventually, the oligomerizing material accumulated in a comparably broad low electrophoretic mobility band.

We imaged the final oligomerization products using negative stain transmission electron microscopy (TEM). The micrographs revealed predominantly circular structures with cone-shaped appearance (Fig. 2B) consisting of 9, 10, 11 , 12, and rarely, 13 copies of wedge subunits, respectively. The extent of heterogeneity seen in the cone oligomers with respect to how many wedges are included per cone is presumably linked to the finite elasticity of the wedge building blocks and their interaction interfaces. These properties may be tuned, if so desired, analogously as previously described with planar ring assemblies. ¹⁴ However, in the present case for the target application, the distribution of cone products covering species ranging from 9 up to 13 wedges appears advantageous for dealing with pleomorphic virus samples. The distribution of cone products seen by TEM explains in part the comparably broad product band in the gel electrophoretic analysis. We also found that in the presence of elevated magnesium concentration such as those used in the gel electrophoresis, the cones have the tendency to stack onto each other (Fig. 12), which explains the smearing and formation of aggregates in the high-magnesium gel electrophoresis such as those shown in Fig. 2A. The cone-to-cone stacking was absent at low magnesium conditions as we show further below.

We observed three key preferred orientations of the cones in the TEM micrographs (Fig. 2B, inset) including cones adsorbed with their bases on the surface (1 ), cones that landed on their vertex (2), and cones that adhered on their lateral facets (3). Vertex-adhered cones had larger diameters and frayed circumferences compared to base-adhered cones containing the same number of wedge building blocks. Presumably, in the vertex-adhered orientation, adhesion forces flatten the cones which then causes the wedges to splay apart. In the based-adhered orientation, the cones remained intact buttressed by their base.

We computed two-dimensional (2D) class averages from TEM micrographs, which revealed several classes corresponding to different views (Fig. 13) and to different cone species. Figure 2C shows exemplarily the class averages obtained for base- adhered cones featuring nine to thirteen wedge subunits, respectively. We measured the diameters from the non-deformed base-adhered particles (1 ) in the respective averaged classes and they closely matched our expectation (Fig. 2D). Accordingly, the C9 cone species had an average inner diameter of 110 nm. The C10 had 126 nm, and largest C13 species had 147 nm. From the 2D class averages we also quantified the relative frequency of occurrence of the different cone species. The most abundant cone was the C10 with a 37 % of the population, followed by C11 (27 %), C9 (20 %), C12 (15 %) and C13 (1 %).

We performed cryo-EM studies of the cones in free-standing ice in order to gain 3D information of the assembled products. The exemplary cryo-EM field of view (Fig. 2E) shows different orientations of partial and fully assembled cones. We determined 3D reconstructions for the C9 and C10 cone species, which confirmed the overall 3D conical shape (Fig. 2F and Fig. 14). The electron density maps of both cone species have elliptical, undulated bases. The ellipticity is more pronounced for the C9 cone map. We measured the lengths of interior short and long axes to be 100 nm and 122 nm for the C9, and 114 nm and 131 nm for the C10 species (Fig. 15). The C9 cone’s cavity was 42 nm deep, whereas the C10’s was shallower (39 nm). The circumferences of the base-adhered cones from negative stain data and in solution cryo-EM reconstructions are in good agreement (Table 8). We assume that the electrostatic interactions between the cones and the carbon surface of the grids used for negative stain TEM leads to a flattening effect and therefore a rounder shape of the rings. It is also possible that surface interaction at the sample-air interface prior to plunge- freezing resulted in deformation of the particles seen in the 3D maps. Reconstructions of subsets of the particle ensemble of the cryo-EM data and multibody refinement and principal component analysis indicate a certain level of flexibility of the cones (Fig. 16), which is desirable for the intended application.

At the salt concentrations present in physiological fluids, DNA origami higher-order assemblies such as those presented in this work would normally dissociate. ¹⁷ The wedge monomers would also be prone to denature due to insufficiently screened internal repulsive electrostatic forces. Physiological environments may also contain nucleases capable of degrading exogeneous DNA molecules by catalyzing the hydrolytic cleavage of phosphodiester bonds in the DNA backbone. ¹⁸ To make our cone assemblies last in in vivo-like conditions, we established a three-step post- assembly stabilization treatment as illustrated schematically in Fig. 3A. The first step utilizes UV-light-induced cross-linking of thymidine bases placed in close proximity within DNA nanostructures. ¹⁹ Through irradiation at a wavelength of 310-nm, the double bonds of adjacent pyrimidines undergo a [2+2] cycloaddition reaction yielding a cyclobutane pyrimidine dimer. To UV cross-link (“UV-point-weld”) the cone assemblies, we placed additional unpaired thymidine bases at the helical interfaces of the wedge-wedge subunit interaction sites (yellow dots in Fig. 3A, B). We tested the efficacy of UV cross-linking of cones as a function of time of exposure to irradiation with a 310 nm light source (Fig. 3C). Once properly UV welded, the cones remained intact when exposed to low Mg ²⁺ concentrations, whereas the non-irradiated or insufficiently irradiated control samples rapidly dissociated into the constituent wedge subunits (Fig. 30, D). The UV-linked cones now appear as five distinct bands in a low ionic strength gel (3 mM MgCh).

To protect the cone assemblies against nuclease-mediated degradation, we utilized the previously described oligolysine-PEG copolymer-based coating ²⁰ followed by glutaraldehyde-crosslinking of this coating ²¹ (Fig. 3A). We treated the UV-point-welded cones with K10PEG5K (N:P ratio of nitrogen in lysine to phosphorus in DNA of 1 :0.6, and 2% (v/v) glutaraldehyde). To test for protection against nuclease activity, we subjected the samples to DNase I (0.001 U/pl, which corresponds to 2.6x of typical blood concentration of DNase I). We analyzed the digestion products using direct imaging with negative stain TEM. When uncoated, the cone assemblies were completely digested after 8 hours of incubation with DNase I, whereas the cones remained stable without obvious structural damage for up to 48 hours when oligolysine- PEG coated and cross-linked with glutaraldehyde (Fig. 3E).

For the intended application to tile and occlude the surface of virus particles, the inward-facing surface of the cones must be functionalized with additional virus-binding moieties. To this end, we introduced single-stranded DNA overhangs (termed ‘handles’) on the wedge subunit’s inner surface that can hybridize with sequence- complementary oligonucleotides modified with the virus-binding moiety of choice. The positioning and the number of handles displayed on the wedge surface may be controlled by design. When using strong virus binders such as antibodies, a rather low density of handles may be sufficient for virus trapping (Fig. 4A); whereas weak and more broadly binding virus binders such as heparan sulfate (HS) polymers ²² may benefit from a higher density of handles to exploit multivalency and avidity effects. To covalently conjugate DNA strands to the virus binders we used sulfo-SMCC linker (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1 -carboxylate) for antibody conjugation, ¹¹ and a copper-free click chemistry approach for HS derivatives. ¹²

With the cone assemblies stabilized and functionalized, we tested the cones’ ability to assemble around viruses with Influenza A/Puerto Rico/8/1934 viruses. Hemagglutinin (HA) and neuraminidase (NA) are the two most abundant proteins on the surface of Influenza A virus particles. We selected an antibody (see materials & methods) that targets a conserved epitope of the stem region withing the HA trimer as the virus- binding moiety and used it with a calculated density of six copies per wedge subunit. The antibody-functionalized cones successfully assembled around Influenza virus particles as we observed by direct TEM imaging (Fig. 4B). The cones adapted and molded around the diversely shaped Influenza particles (Fig. 4C). To gain more detailed information on the extent of 3D surface coverage by selected cone-virus assemblies, we performed negative stain electron microscopy tomography (Fig. 4D). The slices of the exemplary 3D tomogram reveal an Influenza virus particle enclosed by two cones in a sandwich-like assembly. Cones without antibodies did not associate with Influenza viruses (Fig. 17).

To further increase the surface area that will be occluded on virus particles, we designed a spiked cone assembly, in which a second wedge subunit (t2) is assembled on the base of the cone (Fig. 5A, B). The t2 wedge has a bevel angle of 45° and binds to the rim of the t1 wedge via a second set of shape-complementary pattern of protrusion and recesses (Fig. 5A, Fig. 18-20 for cryo-EM validation and S21 -22 for assembly characterization). With the addition of the t2 building block, a single spiked cone assembly has an overall cavity depth and diameter of approx. 125 nm (Fig. 5B). A single copy of a spiked cone thus would in principle be sufficiently large to fully engulf Influenza viruses. Fig. 5C shows exemplarily negative stain TEM micrographs that we acquired of spiked-cone Influenza assemblies. The images reveal the flexibility and the different conformations the spiked cone can adopt. The t2 subunit also incorporated handle positions in its inner surface to place virus binding moieties. Similar to the cone assemblies, also the spiked cone variant successfully formed complexes with the Influenza A/Puerto Rico/8/1934 when functionalized with antibodies, as we saw by TEM imaging (Fig. 5D). Single copies of spiked cones were now sufficient to fully enclose entire virus particles of varying sizes. Negative stain TEM tomography was again used to obtain detailed 3D information. Fig. 5E shows tomogram slices through a 3D tomogram acquired of an exemplary spiked-cone Influenza assembly, revealing clearly that the Influenza virus “guest” sits deep within the cavity of the spiked cone “host”.

To illustrate the modular functionalization with virus-binding moieties, we trapped different virus particles with the spiked cone assemblies using the more broadly binding heparan sulfate (HS) derivative as internal coating. When using 12 copies of HS per wedge subunit, Chikungunya, SARS-CoV-2 and Zika virus-like particles (VLP) were also trapped successfully within the spiked cone assemblies, as we established by direct imaging with negative staining TEM (Fig. 23). Depending on the rigidity of the virus particle, either the cone host or the guest virus particle adapted to one another. For instance, the Zika particles completely flattened out when adhered to the cones, whereas the cones deformed to match the curvature of the rather spherical and apparently more rigid Chikungunya particles.

CONCLUSIONS

We presented cone-shaped DNA origami higher-order assemblies that form efficiently and with high yields from a single building block. In comparison to our previous prototypes which took weeks to assemble, required multiple building blocks, and had inferior yields (<50%), we achieved substantially improved assembly yields of above 80% in one-pot reaction mixtures over the time course of 72 hours. We developed the cone assemblies primarily for trapping and engulfing large and pleomorphic virus particles. To this end, we demonstrated modular functionalization with user-defined virus-binding moieties. In one instance, we used antibodies to engulf Influenza viruses with the cones, with up to 60 antibodies displayed per cone. In another instance, we used heparan sulfate to trap Zika, Chikungunya and SARS-CoV-2 VLPs using cone assemblies displaying up to 120 HS polymer copies per cone. The cone assemblies can deform and adapt to the shape of the trapped virus particles, as we saw here with pleomorphic Influenza virus samples, which is advantageous for our envisioned target application. We have also established a post-assembly stabilization treatment of the cones so that they can persist in low-salt environments and survive the attack of nucleases for at least 48 hours. All DNA components needed for our cones can in principle be biotechnologically mass-produced. ²³ The present work thus contributes to setting the stage for testing the therapeutic potential of a large-virus-engulfing DNA nanoarchitecture in vivo. Beyond trapping large viruses, the cone assemblies, or variants of it, could be of use in artificial light-harvesting antenna complexes, ^{24 25} and as a candidate structure for placement on nanostructured surfaces. ^{26 27}

MATERIALS AND METHODS

Staple strands for origami folding reactions were purchased from Integrated DNA Technologies (IDT) and used with standard desalting purification. SH-modified handle strands were purchased from Biomers at 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.

DNA ORIGAMI DESIGN

The cross-sections of both triangular building blocks t1 and t2 are 3x6 arranged in square lattices of DNA helices.

The DNA origami designs of the t1 and t2 isosceles triangles involve corners of different angles as well as a beveled angle. A schematic representation of the important parameters can be found in Fig. 6, A and B. To create a corner in a DNA origami object, specific deletions are necessary depending on the angle of interest. The length difference in between two DNA double helices (Aa) is dependent on the angle (a) and the distance between the two helices (x) following equation (1 ).

The distance between the two helices (x) is the diameter of a DNA double helix (d). The effective diameter of a DNA double helix is 2.1 nm, ³⁹ but considering that in a DNA origami structure the helices are not tightly packed due to electrostatic repulsion forces, d is averaged to be 2.6 nm. ⁴⁰ Depending on the position of each helix (n), x varies and Aa has to be re-calculated using equations (1 ) and (2). With these design parameters, the DNA helices get shorter the closer they are to the center.

Isosceles triangles have two different angles (a and p) and therefore require two different corner designs. The length differences of the helices at such comers will be different (Aa and Ab), and need to be calculated separately using equations (1 ) and (2). The length of any helix (a _x or b _x ) can be calculated by subtracting Aa/b from the length of the reference helix (ao or bo). Also, a helical rise of 0.34 nm/bp can be used to convert lengths of DNA helices from base pairs to nanometers.

For corner designs, it is important to know the double helix orientation of the DNA strands at the nick position. In order to reach the other side of the nick, the DNA strand facing the outer side of the comer needs to have a single stranded segment (Fig. 7C). When the staple strand (yellow) faces the outer side of the nick, we give it 5 thymidine single stranded bases, whereas when it is the scaffold strand (blue), we only give it one single stranded base.

In order to get assemblies with curvature, the sides of the triangles need to be tilted by a certain bevel angle. Fig. 7D shows a schematic representation of how a comer design looks with a certain beveled angle. By rotating each DNA helix by an angle 0, the original coordinates of a helix (n,m) change from xo.nm and yo.nm to x _n m and y _n m (Fig. 7 A). The new coordinates can be calculated using a two-dimensional rotation matrix (4). All triangles in this work were designed such that the three edges always have the same bevel angle and only different lengths.

Xnml _ [ COS0 Sln01 * [ ^x o,nm with ynnJ 1— sin0 COS0J [yo.nm.

The length differences needed to apply to achieve the desired bevel angle can be calculated using (5.1 ) and (5.2):

If the bevel angle is designed to be pronounced, the resulting assembly will feature a deep cavity at the cost of a smaller cone diameter; whereas if it is less prominent, the product will have shallower depth but display a larger diameter.

The actual values of corner angles (a and 0), beveled angles (0) and lengths of the reference helices (a _x and b _x ) are summarized in Table 2.

Table 4. Design parameters of t1 and t2 referencing Fig. 7. Folding of DNA origami triangular subunits: DNA origami structures were self-assembled (“folded”) in one-pot reaction mixtures containing 50 nM of single-stranded scaffold DNA (M13, 8064 bases) and 250 nM of each staple strand in a standardized “folding buffer” (FoB15) containing 15 mM MgCl2, 5 mM Tris Base, 1 mM EDTA and 5 mM NaCI at pH 8.00. Scaffold M13 was produced as previously described (Supplementary Note 1 for sequence). ²⁸ The folding reactions were subjected to thermal annealing ramps (60 to 44°C with a decrease of 1 °C/h) in a Tetrad (Bio-Rad) thermal cycling device.

Purification of triangle subunits and self-assembly of cones:

All objects were purified using agarose gel extraction (1 .5 % agarose containing 0.5 x TBE and 5.5 mM MgCl2) and centrifuged for 60 min at maximum speed for residual agarose pelleting. Typical subunit concentrations ranged from 5 to 50 nM, while assembly times ranged from 3 to 5 days. Cone assembly proceeded well at a MgCl2 concentration of 25 mM and incubation at 40°C for at least 72 hours. The assembly of the spiked cone with t2 required 40 mM MgCl2 and a longer incubation time (approx. 4 days).

Cones stabilization for in vivo applications:

The assembled cones were UV cross-linked19 for at least 20 min at 310 nm using Asahi Spectra Xenon Light source 300W MAX-303. The cones were incubated in a 0.6:1 ratio of N/P with a mixture of K10-oligolysine and K10-PEG5K-oligolysine (1 :1 ) for 1 h at room temperature as similarly described previously. ²⁰ For chemical cross- linking, appropriate amounts of a 50% glutaraldehyde stock were added for a final concentration of 2% (v/v), incubated for 1 h at room temperature, and filtered with 0.5 ml Zeba spin desalting columns (7K MWCO). Dnase I activity assays were performed at 0.001 LI/pL (2.6-fold increase of blood concentration) and incubated at 37°C for different time points in 1 x PBS buffer containing 10 mM MgCl2. Generation of recombinant antibody:

Sequences of the heavy variable chain and the lambda light variable chain of the broadly reactive monoclonal antibody CR9114 specifically targeting the stem region of the Influenza A and B hemagglutinin (HA) ²⁹ were derived from RCSB protein data bank 4FQI, modified with suitable restriction sites for cloning and ordered as strings from GeneartTM. DNA fragments encoding the variable domain of the heavy and light chain were cloned into a pAbHC or pAbLCJambda vector respectively, both pBR322 based human lgG1 expression vectors. Correct cloning was confirmed by Sanger sequencing performed by MicrosynthSeqlab. Antibodies were expressed in 40 ml HEK293F Expi cells. Cells were grown to 2.5x106 cells/ml at the point of transfection. The transfection uses ThermoFisher ExpiFectamine transfection kit and follows the included protocols. 40 pg DNA (20 pg heavy chain plasmid, 20 pg light chain plasmid) were transfected using 107 pl ExpiFectamineTM. After 16-18 h 200 pl Enhanced and 2 ml Enhanced were added to the transfected cells. Cells were left to express the antibodies for 5 days at 37°C, 8 % CO2 on an incubator shaking at 125 rpm. Supernatant was cleared by centrifugation at 1 ,000 g for 10 min, followed by 4,000 g for 15 min. Cleared supernatant was sterile filtered (0.2 pm milipore steritop filter) and when stored added with 0.05 % NaNs. HiTrap rProtein A FF 1 ml columns were loaded with the supernatant overnight at 4°C at a flowrate of 1 ml/min. Columns were then washed with 50 ml PBS to wash away any unbound leftovers. Antibodies were eluted using 0.1 M Glycine, pH 3.2 and fractionated 4 times in 2.5 ml. Each fraction was immediately neutralized with 1 M Tris/HCI, pH 9 to a final pH of 7.3. Using pD10 columns the buffer was exchanged to PBS. For storage preparation the antibody was concentrated or diluted to the wanted concentration and centrifuged at 14,000 g for 30 min before being sterile filtered (22 pm).

Antibody conjugation to DNA: An oligonucleotide with a sequence complementary to the origami handles (5'- TGCCTAATCTCTACCTACTCTACTGC-3'; SEQ ID NO: 1408) and modified with a thiol group at the 3' end was coupled to the antibody anti-HA CR9114 (100 pg) using a sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1 -carboxylate crosslinker. The product was purified using proFIRE (Dynamic Biosensors). The DNA-modified antibody was added to the assembled and UV-welded cones with 1 :1 stoichiometry to the number of handles and incubated for 1 h at room temperature.

Heparan sulfate conjugation to DNA:

Experimental protocol was as previously described by Monferrer et.al. ¹²

Viruses and VLPs encapsulation:

Pre-assembled and UV-welded cones in 1 x PBS containing 10 mM MgCl2 were mixed with a virus or VLP sample in the appropriate ratio. The samples were incubated at r.t. for 2 h. Usual amounts of sample for TEM analysis range from 5-10 pL total solution at ~ 10 nM triangle origami concentration. Negative stain TEM grids were prepared immediately after the 2 h incubation.

Negative staining TEM:

Samples were incubated on glow discharged (45 s, 35 mA) forrmvar carbon-coated Cu400 TEM grids (Electron Microscopy Sciences) for 90 to 120 s depending on origami and MgCl2 concentrations. Next, the grids were stained for 30 s with 2% aqueous uranyl formate containing 25 mM NaOH. Imaging was performed with magnifications in between 10000x and 42000x in a SerialEM at 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 auto-leveled using Adobe Photoshop. To obtain TEM statistics in an unbiased fashion, automatic grid montages were acquired. For detailed information on selected particles, negative stain EM tomography was used as a visualization technique. The tilt series were performed from -30° to +30° and micrographs were acquired in 2° increments. Tilt series were processed with Etomo (IMOD) to acquire tomograms. ³⁰ The micrographs were aligned to each other by calculating a cross correlation of the consecutive tilt series images. The tomogram is then generated using a filtered back-projection. The Gaussian-Filter used a cutoff between 0.25 and 0.5, and a fall-off of 0.035.

Negative Stain data processing:

We processed the micrographs in CryoSparc ³¹ and estimated the contrast transfer function (CTF) with CTFFIND4. ³² We used a combination of manual picking and TOPAZ auto-picking ³³ and extracted the particles consisting of different numbers of monomers. We subjected the particles to multiple rounds of 2D classification to sort them and to create class-averaged images at increased signal-to-noise ratio. We evaluated the distribution of assemblies via the assignment of particles in certain 2D classes and manual inspection. We measured the dimensions of different types of assemblies based on the 2D class-averaged images data using FIJI. ³⁴

Cryo-grid preparation and cryo-EM image acquisition:

For the triangle DNA constructs, we vitrified each cryo-EM sample with a Vitrobot Mark IV (Thermo Scientific). We applied 4 pl of sample to a glow discharged C-Flat grid (Protochips) (Table 6), blotted, and plunge-froze it using the following Vitrobot settings: temperature of 22°C, relative humidity of 100%, 2-2.5s blot time, -1 blot force. For the cone assemblies we used double blotting consisting of 4 pl sample application, 60 s incubation on the grid, manual blotting, followed by a second round of sample application, semi-automatic blotting and plunge-freezing as described above. We acquired movies consisting of 10-13 frames with a Falcon 3 direct detector (Thermo Scientific) on a Cs-corrected (CEOS) Titan Krios G2 electron microscope (Thermo Scientific) operated at 300 kV using the EPU software (Thermo Scientific) at an accumulated dose of ~50 e/sqA and a magnified pixel size of 2.28 A and 1 .79 A (Table 5). Acquisition with a tilted stage was used to reduce orientation bias of the particles.

Cryo-EM data processing:

We processed the cryo-EM data mostly in the Relion 4 software suite. ^{35 36} For motion- correction of the movies and CTF-estimation we used the Relion implementation and CTFFIND4, ³² respectively. We semi-automatically picked particles using TOPAZ, ³³ extracted the particles, and removed falsely picked grid contaminations damaged particles via multiple rounds of 2D. Using a low-resolution ab-initio initial model created in Relion we addressed structural heterogeneity via 3D classification and reconstructed a 3D-refined map. We applied per-particle motion correction and dose weighting to receive a set of polished particles and reconstructed a 3D-refined map at higher resolution. We post-processed the map by applying a low-resolution mask as well as Fourier shell correlation (FSC) estimation-based low-pass filtering and sharpening using the 0.143 FSC criterium. For the triangle 2 version 1 , we reconstructed the final map including post-processing using CryoSparc. ³¹ We 3D-measured the dimensions of the electron density maps in 3D and rendered images using ChimeraX. ³⁷

Table 5. Estimation of C9 and CIO’s inner diameters.

Table 6. Cryo-EM grid preparation, data acquisition and data processing details.

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