Before use, colloidal solutions of 50- and 80-nm AuNPs were subjected to centrifugation to concentrate 10 times (50-nm AuNPs, 7000 rpm for 10 min; 80-nm AuNPs, 4000 rpm for 10 min). Concentrated colloidal solution (800 μl) of 80-nm AuNPs was mixed with freshly dissolved thiol-modified DNA (100 μM) in a 1:50,000 ratio (1:10,000 for 50-nm AuNPs) in Milli-Q water, and the mixture was incubated for 2 hours at room temperature (300 rpm). Then, 100 μl of phosphate buffer (PB) [100 mM (pH 7.4)] was added to the mixture. After 30 min, we added 10 μl of NaCl solution (2 M) every 20 min for four times and then 20 μl of NaCl solution (2 M) every 30 min for three times. The NaCl concentration was gradually increased to ensure the full coverage of L-AuNPs with thiolated DNA. The final concentration of NaCl was 200 mM, and the mixture was incubated at room temperature (300 rpm) overnight. The AuNP-DNA conjugates were purified by 0.5% agarose gel electrophoresis [running buffer, 0.5× tris-borate-EDTA (TBE); loading buffer, 50% sucrose; 1 hour at a constant 100 V]. Desired bands were cut out, and thiolated DNA–modified AuNP clusters were extracted from the gel using a protocol given by Bellot et al. (36). Freshly prepared, fully covered 50- and 80-nm AuNPs did not precipitate in the 0.6× TAE-Mg2+ buffer [24 mM tris, 12 mM acetic acid, 1.2 mM EDTA, and 7.5 mM magnesium acetate (pH 8.0)]. This high-salt resistance property of fully covered L-AuNPs makes it possible to assemble metamolecules on a DNA origami template.

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