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Abstract

Although for many purposes, low concentrations of DNAorigami aresufficient, certain applications such as cryo electron microscopy,measurements involving small-angle X-ray scattering, or invivo applications require high DNA origami concentrationsof >200 nM. This is achievable by ultrafiltration or polyethyleneglycol precipitation but often at the expense of increasingstructural aggregation due to prolonged centrifugation and final redispersionin low buffer volumes. Here, we show that lyophilization and subsequentredispersion in low buffer volumes can achieve high concentrationsof DNA origami while drastically reducing aggregation due to initiallyvery low DNA origami concentrations in low salt buffers. We demonstratethis for four structurally different types of three-dimensional DNAorigami. All of these structures exhibit different aggregation behaviorsat high concentrations (tip-to-tip stacking, side-to-side binding,or structural interlocking), which can be drastically reduced by dispersionin larger volumes of a low salt buffer and subsequent lyophilization.Finally, we show that this procedure can also be applied to silicifiedDNA origami to achieve high concentrations with low aggregation. Wethus find that lyophilization is not only a tool for long-term storageof biomolecules but also an excellent way for up-concentrating whilemaintaining well-dispersed solutions of DNA origami.