Nanostructures from Synthetic Genetic Polymers

Abstract Nanoscale objects of increasing complexity can be constructed from DNA or RNA. However, the scope of potential applications could be enhanced by expanding beyond the moderate chemical diversity of natural nucleic acids. Here, we explore the construction of nano‐objects made entirely from alternative building blocks: synthetic genetic polymers not found in nature, also called xeno nucleic acids (XNAs). Specifically, we describe assembly of 70 kDa tetrahedra elaborated in four different XNA chemistries (2′‐fluro‐2′‐deoxy‐ribofuranose nucleic acid (2′F‐RNA), 2′‐fluoroarabino nucleic acids (FANA), hexitol nucleic acids (HNA), and cyclohexene nucleic acids (CeNA)), as well as mixed designs, and a ∼600 kDa all‐FANA octahedron, visualised by electron microscopy. Our results extend the chemical scope for programmable nanostructure assembly, with implications for the design of nano‐objects and materials with an expanded range of structural and physicochemical properties, including enhanced biostability.

MALDI-ToF mass spectra of tetrahedron component strands. a) DNA and b) HNA strands used to construct tetrahedra lacking vertex oligos (see Fig S4). Oligos used to construct DNA-tagged tetrahedra, composed of c) DNA, d) FANA, e) 2'F-RNA, f) HNA and g) CeNA. Mass spectra of DNA and all four XNA chemistries are consistent with expected full length products, or full length with single additions through extendase activity. Component strands of DNA tetrahedra [1] can be substituted with equivalents composed of FANA or 2'F-RNA, yielding composite nanostructures, verified by non-denaturing agarose gel electrophoresis (3%, 0.5X TBE).
HNA (and CeNA) versions of any of the four strands could not be combined with DNA; however, composite tetrahedra could be formed with 2'F-RNA and an HNA version of strand A. 2, titling these objects shows little or no parallax motion, revealing that they are 2D objects in the same plane [2] . a) HNA strands (primed with RNA oligonucleotides, which were subsequently removed by alkaline hydrolysis) are used to assemble tetrahedra composed entirely of HNA, as verified by non-denaturing PAGE. b) Degradation (%), as judged by densitometry of samples electrophoresed on non-denaturing agarose gel (3%, 0.5X TBE, stained with GelStar)(inset), is plotted vs. time for all-DNA tetrahedra (black circles) and all-HNA tetrahedra (black triangles). HNA tetrahedra, unlike DNA equivalents, are not degraded but remain stable during prolonged incubation at 37 o C in Hank's Balanced Salt Solution (HBSS) supplemented with 10 % FCS. a) In step 1, a double stranded DNA product comprising the DNA octahedron heavy chain sequence [3] (OHC) (with 5' Cy3 label (magenta star)) is obtained by PCR amplification of a synthetic gene, verified b) by 2% agarose gel electrophoresis (0.5X TBE). In step 2, the PCR product is captured on streptavidin magnetic beads through the (biotinylated) DNA template strand; (unbiotinylated) DNA OHC is denatured and eluted using sodium hydroxide. In step 3, the DNA template strand directs the synthesis of the 1,669 nt FANA OHC (with 5' Cy5 label (cyan star)) using an engineered XNA polymerase (PolD4K [4] ), verified c) by Urea-PAGE (10% acrylamide, 1X TBE). NB: Like RNA, FANA oligonucleotides have a slower PAGE mobility than DNA, although mobility maybe be affected by incomplete denaturation of the extensive heavy chain secondary structure, which is also evident in samples of template DNA.  Alternative models for the XNA (FANA) octahedron obtained form single-particle reconstruction using RELION 1.3 [11] are shown in yellow (surface) and grey (surface and mesh). The volume enclosed in both cases corresponds to 450 Å 3 . The two models are proposed to correspond to two slightly different conformations.
Reagents were obtained from Sigma Aldrich (USA) unless specified otherwise.

XNA synthesis and purification
To prepare component strands [1] for XNA tetrahedra bearing DNA oligonucleotides, XNA syntheses were performed as described previously strands were synthesized using 'RNA_FD' primer, which was hydrolyzed after isolation of ssXNA by incubation in 0.7 N NaOH for 1 hr at 65 o C. XNA strands were purified by Urea-PAGE, as described previously [6] .
To prepare the 1,669nt octahedron heavy chain, the synthesis scheme shown in Fig. S4  and captured with streptavidin beads (1 μg beads / pmol PCR product) as described above.
(+)strand (i.e. DNA octahedron heavy chain) was denatured and eluted using 0.1 N NaOH, then to ice for 2 min. After synthesis, reactions were incubated with streptavidin beads and FANA octahedron heavy chain denatured and eluted using NaOH, as described above.
GNPs coupled to single XNA strands were separated from poly-and unconjugated GNPs using agarose gel electrophoresis (at 4 o C), electro-eluted onto GF/B glass fiber (Whatman, UK) and recovered by filtration through a 0.45 µm Spin-X filter (Corning, USA).

MALDI-ToF mass spectrometry
Oligos were resuspended in water to 1 uM. 0.75 µl samples were spotted onto a maldi target followed by 0.75 µl 3-hydroxypicolinic acid. All mass spectrometric measurements were carried out in positive ion mode on an Ultraflex III TOF-TOF instrument (Bruker Daltonik, Bremen, Germany).
Bands corresponding to correctly-folded octahedra were electro-eluted into TBM supplemented with 30% sucrose in a well cut into the upper layer. Purified octahedra were exchanged into 1X OCT buffer using 3,000 MW cut-off PES spin filters (Sartorius, Germany).

Transmission electron microscopy of XNA structures
In order to image GNP-labeled tetrahedra, samples were deposited onto formvar-coated 400 mesh copper grids (Agar Scientific, UK). Grids were ionized by glow discharge, floated onto sample drops for 5 min, wicked with filter paper to remove excess sample. 'Quasi-3D' [2] images were collected at several holder α tilt angles using a Tecnai T12 electron microscope (FEI, USA) operating at 120 kV with an Orius detector (Gatan, USA) at a nominal magnification of x10,000.
A set of 1,314 particles was assembled by manual picking using boxer, part of the EMAN2 package [10] . The particles were extracted into 200x200 boxes, coarsened, normalized and band-pass filtered between 150 and 25 Å. The 3D classification into 4 classes was carried out in RELION 1.3 [11] using a smooth sphere as starting model and using octahedral symmetry during refinement.
Indeed the analysis of the projection images indicates that views with 4-fold and other views with 2and 3-fold rotational symmetry are present, consistent with the 432 octahedral point group symmetry. Two 3D maps are presented here which were selected based on connectivity between the density regions and overall shape.