Custom-Size, Functional, and Durable DNA Origami with Design-Specific Scaffolds

[Image: see text] DNA origami nano-objects are usually designed around generic single-stranded “scaffolds”. Many properties of the target object are determined by details of those generic scaffold sequences. Here, we enable designers to fully specify the target structure not only in terms of desired 3D shape but also in terms of the sequences used. To this end, we built design tools to construct scaffold sequences de novo based on strand diagrams, and we developed scalable production methods for creating design-specific scaffold strands with fully user-defined sequences. We used 17 custom scaffolds having different lengths and sequence properties to study the influence of sequence redundancy and sequence composition on multilayer DNA origami assembly and to realize efficient one-pot assembly of multiscaffold DNA origami objects. Furthermore, as examples for functionalized scaffolds, we created a scaffold that enables direct, covalent cross-linking of DNA origami via UV irradiation, and we built DNAzyme-containing scaffolds that allow postfolding DNA origami domain separation.