DNA-origami-directed virus capsid polymorphism | Nature Nanotechnology

DNA origami folding and purification

The DNA origami constructions (6HB, 24HB, 60HB, 13HR and nanocapsule) had been folded in a one-pot response by steadily reducing the temperature utilizing a Proflex 3 × 32-well PCR system (Thermo Fisher). The scaffold strands (p7249, p8064 and p7560 variants of single-stranded M13mp18) had been bought from Tilibit Nanosystems and the staple strands from Built-in DNA Applied sciences. To make sure excessive folding yields of DNA origami, structure-specific optimized situations concerning each the annealing procedures and the buffer alternative (‘folding buffer’, FOB) are used (Supplementary Word 20).

Buffer change for DNA origami

The purified DNA origami constructions had been transferred into 6.5 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer supplemented with 2 mM NaOH (HEPES-NaOH, pH 6.5) earlier than complexation with CCMV CPs. The buffer change was carried out by spin-filtration⁵⁹ utilizing 100 kDa molecular weight cut-off (MWCO) centrifugal filters (Amicon), which had been washed earlier than use by centrifuging with 400 μl of HEPES-NaOH buffer for five min at 14,000g. Subsequently, equal volumes of DNA origami answer and HEPES-NaOH buffer had been added into the filter system and the centrifugation was continued for 10 min at 6,000g. A quantity of HEPES-NaOH equal to 2.09× the preliminary quantity of origami answer was then added, and the centrifugation step was repeated. The pattern was collected by inverting the filter and centrifuging for two.5 min at 1,000g.

Isolation of CCMV CPs

The CPs had been remoted from intact CCMV (for virus preparation, see Supplementary Word 21). Briefly, the virus particles had been dialysed in a single day towards 50 mM Tris–HCl, 500 mM CaCl₂ buffer, pH 7.5 supplemented with 1 mM dithiothreitol (DTT) utilizing Slize-A-Lyzer Mini Dialysis cups (3.5 kDa MWCO, Thermo Scientific). The RNA was pelleted in a centrifugation step at 4 °C utilizing 21,100g for six h, and the recovered supernatant was dialysed in a single day towards ‘clear buffer’ that accommodates 50 mM Tris–HCl, 150 mM NaCl at pH 7.5 supplemented with 1 mM DTT (tailored from ref. ⁶⁰). The focus of the proteins was decided primarily based on their absorbance at 280 nm (extinction coefficient, 23,590 M⁻¹ cm⁻¹) utilizing a BioTek Eon Microplate Spectrophotometer (2 μl pattern, Take3 plate).

AGE

AGE was used to check the binding interplay between the proteins and the origami constructions by monitoring the shift in electrophoretic mobility. Moreover, the intactness of the origami constructions after folding and purification, and through DNase I digestion, was analysed by gel electrophoresis. To this finish, samples (volumes starting from 10 to 32 μl) supplemented with 6× gel loading dye (40% sucrose with out dye for samples from digestion research) had been run in a 2% (w/v) agarose gel (1 × Tris–acetate–ethylenediaminetetraacetic acid (TAE) buffer, 11 mM MgCl₂) for 45 min at 90 V in 1 × TAE buffer supplemented with 11 mM MgCl₂. For staining, ethidium bromide (EtBr) at a last focus of 0.46 μg ml⁻¹ was used and the DNA was visualized underneath ultraviolet mild utilizing a GelDoc XR+ system (Bio-Rad).

Complexation of DNA origami and CCMV CPs

The complexation between CPs and DNA origami was carried out at a last origami focus of 4 nM (10 μl samples). The origami was added in a 1:1 quantity ratio to the protein answer that had been diluted within the ‘clear buffer’. Relying on the required protein extra, ε, which describes the molar ratio between CP to DNA origami, protein options starting from 0 to 60 μM (akin to ε = 0–15k) had been ready. The NaCl focus was adjusted to 150 mM, leading to a complexation buffer containing 3.25 mM HEPES-NaOH, 25 mM Tris–HCl, 150 mM NaCl and 0.5 mM DTT. The complexation was carried out at 4 °C for not less than 1 h and subsequently analysed utilizing AGE and TEM.

DNase I digestion assays

To review the safety impact of the CP coating towards degradation of the origami constructions by DNase I, 2 μl of DNase I inventory (starting from 0 to 500 KU ml⁻¹) was added to 16 μl of the pattern. Moreover, CaCl₂ and MgCl₂ concentrations had been adjusted, leading to a last response quantity of 20 μl containing 3.2 nM DNA origami, 2.6 mM HEPES-NaOH, 20 mM Tris–HCl, 120 mM NaCl, 0.4 mM DTT, 1 mM CaCl₂ and 5 mM MgCl₂. The samples are incubated at 37 °C for 15 min (6HB) and 60 min (24HB). Earlier than analysing the result by AGE, samples complexed with CPs had been disassembled utilizing heparin sodium salt as a aggressive binding agent (last concentrations of 1.5 μM for 6HB-2k and 24HB-2.5k and 82 μM for 6HB-10k and 24HB-10k; Supplementary Word 14).

RNA–DNA origami folding and purification

For the RNA–DNA hybrid origami (RNA-6HB), EGFP mRNA (CleanCap EGFP mRNA, TriLink Bio Applied sciences, L-7601) was used because the scaffold. In a one-pot response, the 996-nt-long mRNA scaffold was thermally annealed with 29 staple strands (bought from Built-in DNA Applied sciences, see Supplementary Word 22) into a brief 6HB construction utilizing a Proflex 3 × 32-well PCR system (Thermo Fisher). The construction is designed to include two scaffold crossovers and has a helical pitch of 11 bp per flip. For the folding response, the mRNA and the staples had been diluted into 1 × FOB containing 1 × TAE pH 8.4, 5 mM MgCl₂ and 1 mM NaCl reaching last concentrations of fifty nM and 500 nM, respectively. The response combination was incubated at 55 °C for 15 min⁶¹ and cooled down by putting it on ice for not less than 10 min earlier than storage at 4 °C. To validate the folding, 4 staple strands had been exchanged with staple strands containing a 3′ overhang (labelled with F, Supplementary Desk 2). A fluorophore-containing attachment strand (ATTO590, Built-in DNA Applied sciences), which was added to the folding combination in 10× extra per attachment web site, can then be built-in into the construction by hybridization with the staple overhangs.

The folded constructions had been purified from extra staple strands by spin-filtration. To this finish, the filter (100 kDa MWCO, Amicon) was washed with 400 μl of 1 × FOB by centrifugation at 14,000g for five min, adopted by two-times addition of 40 μl RNA-6HB along with 40 μl of 1 × FOB. After a centrifugation step at 6,000g for 10 min, 80 μl of 1 × FOB was added and the centrifugation continued (6,000g, 10 min). This washing step was repeated in complete 3 times earlier than the pattern was recovered by inverting the filter right into a clear tube (1,000g, 2.5 min). The focus was decided by measuring the absorbance at 260 nm (extinction coefficient, 1.29 × 10⁷ M⁻¹ cm⁻¹), and the profitable folding was decided by AGE (3.5 % (w/v) gels, visualization underneath ultraviolet mild (EtBr channel) and pink mild (A647 channel), ChemiDoc MP system, Bio-Rad), AFM and TEM.

Complexation of RNA-6HB origami and CCMV CPs

For the complexation, purified RNA-6HB origami in 1 × FOB was combined with CCMV capsids in ‘clear buffer’ in a 1:1 ratio at a last hybrid origami focus of seven.5 nM. This ends in a complexation buffer containing 45 mM Tris, 75.5 mM NaCl, 10 mM acetic acid, 2.5 mM MgCl₂, 0.5 mM DTT and 0.5 mM EDTA. The samples had been incubated at 4 °C for not less than 1 h earlier than evaluation with AGE and TEM.

Complexation of DNA origami and NoV CPs

NoVLPs had been ready as reported by Lampinen et al.⁶² and saved in 1 × phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 1.8 mM KH₂PO₄, pH 7.4); nonetheless, right here SpyTag003 (ref. ⁶³) has been fused to the C-terminus of the VP1 from the NoV pressure Hu/GII.4/Sydney/NSW0514/2012/AU. The particles had been high quality managed with dynamic mild scattering for particle formation, sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein purity and the residual dsDNA was measured. For the complexation with DNA origami, DNA origami was current within the pattern throughout each disassembly and reassembly of the VLPs. To this finish, the origami constructions had been transferred into deionized water utilizing spin-filtration (as described above). The DNA origami was combined with the NoVLPs at completely different concentrations in a 1:4 (v/v) ratio, leading to a last origami focus of 6 nM (30 μl samples). The samples had been transferred into 3.5 kDa MWCO dialysis cups (Slize-A-Lyzer, Thermo Scientific) and dialysed in a single day at 4 °C towards 50 mM Tris–HCl, pH 8.9. For reassembly, the samples had been, in a second step, dialysed in a single day at 4 °C towards 100 mM sodium phosphate buffer, pH 6.0, equally as reported by White et al.⁶⁴ The complexation throughout disassembly and meeting of the NoVLPs was analysed by AGE and TEM.

Complexation of DNA origami and SV40 CPs

The SV40 main CP VP1 (abcam, ab74565) was disassembled and reassembled (tailored from ref. ⁵⁰) by dialysing the assembled VLPs in PBS towards 20 mM Tris, 2 mM DTT, 5 mM EDTA and 50 mM NaCl, pH 8.9 for two h at 4 °C (3.5 kDa MWCO, Slize-A-Lyzer, Thermo Scientific), after which the EDTA focus was decreased by an extra dialysis step at 4 °C for two h towards 20 mM Tris, 2 mM DTT, 2 mM EDTA and 50 mM NaCl, pH 8.9. The focus was decided primarily based on the absorbance at 280 nm (VP1 extinction coefficient, 32,890 M⁻¹ cm⁻¹). The DNA origami was transferred into 100 mM HEPES buffer, pH 7.2, supplemented with 125 mM NaCl by spin-filtration (as described above). The proteins had been combined with the DNA origami in 1:1 (v/v) ratio to succeed in last concentrations of 0–20 μM and a couple of nM, respectively, and the samples had been incubated for twenty-four h at room temperature earlier than evaluation utilizing AGE and TEM.

Complexation of DNA origami and MPyV CPs

For the complexation of VP1 capsomers (for recombinant expression and purification, see Supplementary Word 23) and DNA origami, the origami constructions had been first transferred into 40 mM Tris buffer, pH 8.0, supplemented with 20 mM acetic acid, 2 mM EDTA and 12 mM MgCl₂ utilizing spin-filtration (see above). Relying on the specified extra of proteins, ε, the capsomers had been diluted into ‘storage buffer’, containing 40 mM Tris, 200 mM NaCl, 1 mM EDTA, 5% (v/v) glycerol and 5 mM DTT, pH 8.0. For the complexation, VP1 capsomers had been diluted in a ratio of 1:20 within the origami answer, leading to a last origami focus of 0.75 nM (30 μl samples) and a complexation buffer containing 40 mM Tris, 19 mM acetic acid, 1.95 mM EDTA, 11.4 mM MgCl₂, 10 mM NaCl, 0.25% (v/v) glycerol and 0.25 mM DTT, pH 8. The complexation response was incubated at 4 °C in a single day earlier than evaluation with AGE and TEM.

AFM

A 20 μl droplet of 10 nM RNA-6HB origami answer (MgCl₂ focus adjusted to 12.5 mM) was deposited on a freshly cleaved mica substrate (Electron Microscopy Sciences) for 1 min, adopted by three washing steps with 100 μl deionized water that was instantly blotted away. The pattern was dried underneath a gradual nitrogen stream and imaged instantly after pattern preparation. AFM photos had been acquired in air utilizing ScanAsyst in Air Mode along with ScanAsyst-Air probes (Bruker) on a Dimension Icon AFM (Bruker). Picture processing was carried out in NanoScope Evaluation v.1.90 (Bruker).

TEM

Plain DNA origami samples (4 nM) had been ready by incubation of a 3 μl droplet for 3 min on a plasma cleaned (20 s oxygen plasma flash, Gatan Solarus) Formvar carbon-coated copper grid (FCF400Cu, Electron Microscopy Sciences), which was subsequently blotted towards filter paper and detrimental stained. For CCMV-CP-complexed samples (4 nM DNA origami), a 3 μl droplet was deposited on the grid for 1.5 min. After blotting towards filter paper, the grid was immersed in a ten μl droplet of complexation buffer (3.25 mM HEPES-NaOH, 25 mM Tris–HCl, 150 mM NaCl, 0.5 mM DTT) for five s. For samples with DNA origami concentrations ≤2 nM (for instance, complexation with SV40, MPyV), and for samples containing RNA-6HB (7.5 nM origami focus), the droplet dimension was elevated to five μl and the incubation time prolonged to five min. Unfavorable staining⁶⁵ was carried out by first immersing the grid in a 5 μl droplet of aqueous 2% (w/v) uranyl formate answer (supplemented with 25 mM NaOH for pH adjustment), which was instantly blotted away. This step was adopted by an immersion in a 20 μl droplet, which was incubated on the grid for 45 s. After the ultimate blotting step, the samples had been left to dry for not less than 20 min earlier than imaging was carried out on a FEI Tecnai 12 Bio-Twin microscope at an acceleration voltage of 120 V.

Cryo-EM

The samples for cryo-EM had been ready utilizing a vitrification equipment (Vitrobot, Thermo Fisher Scientific). The origami concentrations within the complexed samples had been 90 nM for 6HB-2k, 84 nM for 24HB-2.5k, 18 nM for 6HB-10k and 21 nM for 24HB-10k, leading to complete CP concentrations of 180 μM and 210 μM for complexed 6HB and 24HB samples, respectively. A 3 μl aliquot of the complexed origami pattern was deposited on a plasma-cleaned (50 s, Harrick Plasma PDC-002-EC instrument) holey carbon-coated grid (copper 200 mesh R1.2/1.3, Quantifoil). After a 1 min incubation, extra liquid was blotted for 10 s at 100% relative humidity and 6 °C, adopted by plunging the grid into liquid ethane. The grids had been saved in liquid nitrogen. Information had been collected at liquid nitrogen temperature in a Talos Arctica transmission electron microscope (Thermo Fisher Scientific) operated at 200 kV, utilizing a Falcon III direct electron detector (Thermo Fisher Scientific). A magnification of 150,000× was used, leading to a calibrated pixel dimension of 0.96 Å. The info assortment parameters are listed in Supplementary Desk 4 (Supplementary Word 24).

Single-particle reconstruction

Cryo-EM information had been processed utilizing CryoSPARC 3.3.2 (Structura Biotechnology) until acknowledged in any other case. Distinction switch perform parameters had been estimated utilizing CTFFIND4 (ref. ⁶⁶). Segments alongside filaments had been outlined utilizing the Filament Tracer perform. Helical symmetry parameters had been estimated initially from 2D class averages utilizing Python-based Helix Indexer⁶⁷. The construction and helical symmetry parameters had been refined utilizing Helix Refine perform and non-uniform refinement on motion-corrected helix segments. To find out the helical symmetry parameters of the 6HB-10k outer layer, a second 2D classification run was carried out after subtracting the contribution of the inside layer utilizing the Particle Subtraction perform. The Helix Refine was run on the subset of particles that confirmed a transparent second layer, utilizing the decided symmetry parameters as preliminary estimates. Reconstructions had been sharpened by making use of an advert hoc B-factor of −300 Ų. The reconstructions had been averaged in actual house by imposing the helical symmetry parameters on the central, most ordered a part of the map (50% of the quantity) in Bsoft⁶⁸.

For modelling the construction of the capsomer, CP monomer (PDB:1cwp) was fitted within the 6HB-2k reconstruction within the six positions of the hexamer as inflexible our bodies in UCSF ChimeraX 1.3 (ref. ⁶⁹). The atomic mannequin was refined towards the density utilizing ISOLDE 1.3 (ref. ⁷⁰) and Phenix 1.19 (ref. ⁷¹). To create atomic representations of the filaments, symmetry copies of the hexamer had been created in ChimeraX. To visualise the position of CP hexamers and pentamers within the cap, the caps of the 6HB-2k filament had been manually picked within the micrographs. The cap construction was refined utilizing the Helix Refine perform omitting symmetrization, as this allowed limiting the lean angle of the caps near facet views. Reconstruction of the cap was filtered to its native decision utilizing Native Filter. The hexamer atomic mannequin and beforehand decided pentamer construction (extracted from PDB:1cwp after making use of icosahedral symmetry) had been fitted as inflexible our bodies in ChimeraX 1.3. Information-processing parameters are given in Supplementary Desk 4. Mannequin refinement and validation parameters are proven in Supplementary Desk 5.

SAXS

The samples for SAXS had been ready at origami concentrations of 165 nM (6HB, akin to a disassembled CP focus of 330 μM) and 180 nM (24HB, akin to a disassembled CP focus of 450 μM) and sealed inside a 1.5-mm-diameter glass capillary. The measurements had been carried out utilizing a Xenocs Xeuss 3.0C system geared up with a GeniX 3D copper microfocus supply (wavelength λ = 1.542 Å) and an EIGER2 R 1M hybrid pixel detector at a pattern–detector distance of 1,100 mm. Information acquisition was carried out for 3 × 3 h per pattern. To acquire the 1D SAXS information, the 2D scattering information had been azimuthally averaged. The magnitude of the scattering vector q is given by (q,=,4uppi sin theta /lambda) with 2θ being the scattering angle. Information therapy included averaging of the triplicate 2D information of every pattern, background subtraction from the complexation buffer (3.25 mM HEPES-NaOH, 25 mM Tris–HCl, 150 mM NaCl, 0.5 mM DTT) and a type issue was fitted to a cylinder (6HB, 24HB), spheres (T = 3 icosahedral CPs assemblies) and a core–shell cylinder (6HB-2k, 24HB-2.5k) utilizing SasView software program. A Debye–Anderson–Brumberger mannequin was added to account for the background.