Self-Assembling DNA Nanostructures with caDNAno

Using an open source program, caDNAno^{53 (link)}, all structures used here were designed on the honeycomb lattice with a M13mp18 scaffold strand (7249-nt-long, GUILD, www.guildbioscience.com). Sequences of staple strands for the structures were exported from the caDNAno (Supplementary Tables 1–5) and they were synthesized from Bioneer (www.bioneer.co.kr). A folding mixture consists of 20 nM concentration of scaffold DNA, 100 nM concentration of each staple strand, 1 × TAE buffer (40 mM Tris-acetate and 1 mM EDTA, Bioneer) and 20 mM of MgCl₂ (Sigma-Aldrich, www.sigmaaldrich.com). The annealing process for self-assembly of DNA strands was performed by establishing temperature gradients from 80 to 60 °C with a rate of −0.25 °C/min and from 60 to 45 °C at a rate of −1 °C/hr in a thermocycler (T100, Bio-Rad, www.bio-rad.com). Excessive staple strands were removed through five buffer exchange procedures^{35 (link)} at 5 krcf during 8 min and concentration of structures was adjusted using the same buffer used in folding (1 × TAE and 20 mM of MgCl₂). Concentrations of folded structures were measured using a Nanodrop One UV spectrophotometer (Thermo Fisher Scientific, www.thermofisher.com). Purified structures were stored at −4 °C in a refrigerator.