Illustra g 50

Cy5-labeled 10-helix DNA nanotubes composed of 40 single-stranded DNA tiles with 14- or 28-nm spacing between single-stranded protein binding handles were prepared with biotin strands for surface attachment using an annealing protocol as previously described (34 (link)). The nanotube DNA handles at 14 or 28 nm are designed to anneal to DNA strands bound to a SNAP protein encoded with the GFP nanobody. The SNAP protein binds benzyl-guanine-treated DNA oligos. To prepare the benzyl-guanine oligo, benzyl-guanine NHS ester (BG-GLA-NHS; New England Biolabs, Ipswich, MA) was covalently linked to C6-amine oligonucleotides (with or without Cy3 modification, C6-amine-Cy3-a′ or C6-amine-a′). This was accomplished by incubating 0.17 mM C6-amine-a′ or C6-amine-b′ with 11.6 mM BG-GLA-NHS in 0.1 M sodium borate, pH 8.5 at 37°C for ∼4 h with rotation. The BG-labeled oligonucleotide was then purified twice through Illustra G-50 micro columns (GE Healthcare, Buckinghamshire, UK) equilibrated in 2 mM Tris, pH 8.5, since the primary amine reacts with any unreacted benzyl-guanine. The final BG-labeled oligonucleotide concentration was determined by measuring Cy3 absorbance (for BG-Cy3-a′ or BG-Cy3-b′) or by estimating the concentration of single-stranded DNA (for BG-a′ or BG-b′) using a NanoDrop spectrophotometer (NanoDrop One^C, Thermo Fisher, Waltham, MA).