Reusing excess staple oligonucleotides for economical production of DNA origami.

DNA origami has enabled the development of responsive drug-delivery vehicles with precision features that were previously not attainable in bionanotechnology. To reduce the costs of creating therapeutic-scale amounts of DNA origami that need to bear costly modifications with high occupancy, we reused the excess staple oligonucleotides that are left over from the folding process to fold additional origami. We determined that a DNA origami can be successfully folded with up to 80% cost savings by cyclic recovery and reuse of excess staple strands. We found evidence that higher-quality staple strands are preferentially incorporated into origami, consistent with past reports, and therefore are preferentially depleted from the free-strand pool. The folding of DNA origami with staple strands that were reused up to 11 times was indistinguishable by our panel of assays versus a control folded with new strands, so long as the reused oligonucleotides were replenished each cycle with a small excess of fresh strands. We also continued to observe a high degree of cargo loading [e.g. oligo with PS backbone modification (CpG), fluorophores (Cy5 or Cy3), smaller nanostructures (nanocube), etc.] on the origami with each folding cycle. By recovering, reusing, and replenishing excess staple oligonucleotides, it is possible to significantly lessen production costs to create well-formed origami, which is useful to allow more therapeutic designs to be tested.