Fabrication of DNA Nanostructures

The design-specific staple strands were purchased from IDT Technologies in 250 μM scale. The sequence of the staple strands and the design is reported in the Supplementary Information. The p7308 scaffold strand was produced from M13 phage replication in Escherichia coli and was endotoxin-purified using Triton X-114 as described previously13 (link).
For the synthesis of DNs, 10 nM p7308 scaffold was mixed with tenfold excess of staples in TE buffer (5 mM Tris and 1 mM EDTA) containing 10–20 mM MgCl₂. The amount of MgCl₂ varied with the structure: DN1 (10 mM), DN2 (6 mM), DN3 (10 mM), DN4 (10 mM), DN5 (10 mM), DN6 (12 mM), DN7 (14 mM), DN8 (12 mM) and DN9 (6 mM). The solutions were subjected to a thermal annealing ramp on a Tetrad 2 Peltier thermal cycler (Bio-Rad) according to the following schedule: incubate at 65 °C for 15 min, decrease to 50 °C, incubate at 50 °C for 6 h 30 min and decrease to 40 °C at 6 h 30 min °C⁻¹. The quality of folding was analysed by AGE. Solutions of folded DN were concentrated tenfold using a 30k MWCO Amicon Ultra centrifugal filter device (Millipore) and then purified by glycerol gradient ultracentrifugation30 (link). The 45% and 15% glycerol solutions were made in 1 × TE buffer containing the same levels of MgCl₂ as required for folding. The glycerol fractions containing nanostructures was concentrated and buffer exchanged to remove glycerol using a 30k MWCO Amicon Ultra centrifugal filter device. Following purification, the stock solution was diluted appropriately for TEM imaging to verify quality. The stock concentration was determined by ultraviolet absorbance at 260 nm on a Nanodrop spectrophotometer (Thermo Scientific), assuming that A₂₆₀=1 for 50 μg ml⁻¹ DNs. Stock solutions were stored at 4 °C until use.