Uranyl formate

PEG precipitation: Self-assembly reaction mixtures at 20 mm MgCl₂ were mixed 1:1 (v/v) with precipitation buffer containing 15 % PEG 8000 (w/v) (Ph.Eur.), 5 mm Tris, 1 mm EDTA, and 505 mm NaCl (all chemicals from Carl Roth, Karlsruhe, Germany). The solution was mixed by tube inversion and spinned at 16 000 g, at room temperature (RT) for 25 min using a microcentrifuge (Eppendorf 5420, Hamburg, Germany). The supernatant was removed using a pipette. The pellet was dissolved in target buffer as indicated for each set of experiments and incubated for approximately 20 h at RT or 30 °C.
DNA object self-assembly: Structures were designed using caDNAno v0.2.19 (link) DNA scaffold strands of 7249, 7560, 7704, and 8064 bases length derived from the genome of bacteriophage M13 were used for assembly reactions.2b (link) Staple oligonucleotide strands were prepared by solid-phase chemical synthesis (Eurofins MWG, Ebersberg, Germany, HPSF grade). Production of DNA objects was accomplished in one-pot reaction mixtures containing scaffold DNA at a concentration of 20 nm (default) or 50 nm (pointer in Figure S1, RR in Figures 3 and S8, 24 hb, 42 hb, 100 hb), staple DNA oligonucleotides at 200 nm each, and 5 mm TRIS, 1 mm EDTA, 20 mm MgCl₂, and 5 mm NaCl (pH 8). The reaction mixtures were subjected to a thermal annealing protocol using TETRAD (Biorad) thermal cycling devices. The mixtures were first incubated at 65 °C for 15 min and then annealed from 60 to 40 °C in steps of 1 °C per 2–3 h. The reaction products were stored at RT.
Agarose gel electrophoresis: Electrophoresis of the folded DNA objects was carried out in 2 % agarose gels containing electrophoresis buffer (1 mm EDTA, 44.5 mm Tris base, 44.5 mm boric acid, and 11 mm MgCl₂, pH 8.4). The samples were electrophoresed for two hours at 70–90 V in a water-cooled gel box filled with electrophoresis buffer. The gels typically contained ethidium bromide at a concentration of 1 μm. The agarose gels were scanned using a Typhoon 9500 FLA laser scanner (GE Healthcare) at a resolution of 50 μm/px (ethidium bromide: excitation at 535 nm, emission> 575 nm; fluorescein: excitation at 473 nm, emission 520–540 nm) to give 16-bit tif image files, which we analyzed using ImageJ64 V1.47 (U.S. National Institutes of Health). Cross-sectional lane intensity profiles were computed by averaging over grayscale values within a 20–75 pixel wide box drawn over the lane of interest. After linear background correction, the regions of interest were quantified by integrating the area under the peaks. Yields were estimated by comparing the intensity of bands of interest for treated versus untreated samples.
Negative-staining TEM: Samples were adsorbed on glow-discharged formvar-supported carbon-coated Cu400 TEM grids (Science Services, Munich) and stained using a 2 % aqueous uranyl formate solution containing 25 mm sodium hydroxide. Imaging was performed using a Philips CM100 EM operated at 100 kV. Images were acquired using an AMT 4 Megapixel CCD camera. Micrograph scale bars were calibrated by imaging 2D catalase crystals and using the lattice constants as length reference. Imaging was performed at ×28 500 magnification.

Carbon-coated copper grids (Electron Microscopy Sciences, #CF400-Cu) were glow discharged at 30 mA for 30 s. A 4 μL aliquot of a mMIB1 full length sample (0.02 mg/mL) was applied to the grid. After incubating for 1 min, the grid was washed twice with 1.5% Uranyl formate followed by staining with 1.5% Uranyl formate for 2 min. The grids were imaged using a 120 kV Tecnai T12 (Thermo Fisher Scientific) microscope. Images were recorded using an Ultrascan 895 CCD camera (Gatan).