Reusing excess staple oligonucleotides for economical production of DNA origami

ABSTRACT 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 therapeutics-scale amounts of DNA origami that need to bear costly modifications with high occupancy, we reused the excess staple oligonucleotides that are leftover 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 eleven 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 observed a high degree of incorporation of guests on the DNA origami. 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.