AFM images of information-carrying DNA origami nanostructures before and after 40 freeze–thaw cycles

Twist-corrected rectangular DNA origami nanostructures (DONs) containing five biotin (Bt) modifications were exposed to 40 freeze-thaw (F/T) cycles (RT <-> −25 °C or −80 °C) using two strategies. Glycerol was added to achieve final concentrations of 0 mM, 10 mM, and 100 mM. Strategy I. Streptavidin (SAv) was freshly prepared from a 1 g L-1 stock solution in HPLC-grade water and added to the DON samples (in 1× TAE + 10 mM MgCl2) to reach a final SAv concentration of 500 nM, corresponding to a tenfold molar excess over the total Bt sites. The samples were incubated for 30 min at room temperature and subsequently subjected to F/T cycling. Afterwards, 10 µL of DON/SAv solution were deposited on freshly cleaved mica and subsequently covered with 90 µL of 1× TAE + 10 mM MgCl2 to reach a final DON and SAv concentration of 1 nM and 50 nM, respectively. The sample was incubated for 5 min to allow adsorption, after which the surfaces were rinsed three times with 3 mL of HPLC-grade water to remove unbound material and dried under argon (1 bar) prior to AFM imaging. Strategy II. F/T cycling of DONs (in 1× TAE + 10 mM MgCl2) was performed in the absence of SAv. After F/T cycling of , 10 µL of DON solution were deposited on freshly cleaved mica and subsequently covered with 90 µL of 1× TAE + 10 mM MgCl2 to reach a final DON concentration of 1 nM. After 5 min adsorption, the surface was rinsed three times with 3 mL HPLC-grade water and dried under argon (1 bar). 99 µL of fresh 1× TAE + 10 mM MgCl2 were then applied to the mica surface, after which 1 µL of 5 µM SAv solution was added to reach a final concentration of 50 nM. The sample was incubated for 30 min at room temperature, rinsed again three times with 3 mL HPLC-grade water, and dried under argon (1 bar). Each sample (200 µL) was sealed in polypropylene PCR tubes and stored at −25 °C. For F/T cycling, each cycle consisted of freezing at −25 °C for at least 20 min followed by 20 min thawing at room temperature. A total of 40 cycles were performed. For one additional experiment using strategy II, F/T cycling was performed at −80 °C, with each cycle consisting of freezing at −80 °C for at least 8 min followed by 10 min thawing at room temperature. All samples were imaged in air using a Bruker Dimension Icon operated in ScanAsyst mode with ScanAsyst-Air cantilevers. Images were recorded at a scan size of 2 × 2 µm² with 1024 × 1024 pixels and a line rate of 1 Hz.