Stability of Information‐Carrying DNA Origami Nanostructures During Repeated Freeze–Thaw Cycles

<jats:p>Encoding information in molecular arrangements on DNA origami nanostructures (DONs) provides the basis for novel concepts in molecular data storage and computing. To preserve their integrity over long timescales, the information‐carrying DONs are often stored in a frozen state. Here, we investigate the effect of repeated freeze–thaw (F/T) cycles on the structural and functional integrity of DONs carrying biotin (Bt) modifications. Streptavidin (SAv) binding is used to visualize the stored information by atomic force microscopy (AFM) before and after 40 F/T cycles. Two strategies are compared by F/T cycling of (I) SAv‐bound DONs and (II) SAv‐free DONs that are exposed to SAv directly before AFM imaging. Our results reveal that while the DONs retain their overall shape, F/T cycling induces a small amount of damage, leading to slightly reduced SAv binding. Adding glycerol at mM concentrations efficiently protects the DONs and restores the original SAv binding yields. Nevertheless, SAv exposure after F/T cycling leads to slightly higher and more consistent SAv binding yields and a lower background of nonspecifically adsorbed SAv compared to Strategy I. This makes information readout by AFM more efficient and renders Strategy II more convenient for long‐term storage of information‐carrying DONs with repeated information readout.</jats:p>