Staple strands

For the preparation of DNA origami triangles [20 (link)] and six-helix bundles (6HBs) [28 (link)], the 7249 nt M13mp18 scaffold and about 200 staple strands (Metabion international AG, Planegg/Steinkirchen, Germany) were annealed at a molar ratio of 1:10 in 1 × TAE buffer (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) supplemented with 10 mM MgCl₂ (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) by gradually decreasing the temperature from 80 °C to RT over 1.5 h in a Primus 25 advanced thermocycler (PEQLAB, Erlangen, Germany). The samples were purified using Amicon Ultra-0.5 mL spin filters with 100 kDa molecular weight cut-off (Merck KGaA, Darmstadt, Germany). The concentrations of the obtained DNA origami solutions were determined using an Implen Nanophotometer P330 (Implen GmbH, München, Germany) and adjusted to the desired values.
Genomic dsDNA from salmon testes (Thermo Fisher GmbH, Kandel, Germany) was used as a control with similar GC content as the assembled DNA origami nanostructures [29 (link),30 (link)] but no defined length or secondary structure, in order to distinguish between duplex- and superstructure-specific effects. It was dissolved in 1 × TAE buffer with 10 mM MgCl₂ and the concentration (in bp) adjusted using an Implen Nanophotometer P330 (Implen, München, German).

Immediately upon delivery, the freshly synthesized staple strands (Metabion, Planegg, Germany), dissolved in pure water at 100 µM concentrations, were mixed at equal concentrations to yield the complete staple mixture. This stock solution was then divided into 150 µL aliquots and stored at −20 °C in the dark.