DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features1,2,3,4,5,6,7,8,9,10,11,12. These structures are customizable in that they can be site-specifically functionalized13 or constructed to exhibit machine-like14,15 or logic-gating behaviour16. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials3,16,17,18,19,20,21,22, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production23; the shorter staple strands are obtained through costly solid-phase synthesis24 or enzymatic processes25. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising ‘cassettes’, with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.
We thank D. Maslak (TUM Research Center for Industrial Biotechnology, Technical University of Munich) for cost estimation in pilot-scale production at the TUM Pilot Plant for Industrial Biotechnology. We thank T. Gerling, E. Meier, M. Schickinger and J. Funke for discussions. This project was supported by European Research Council starting grant 256270, the Deutsche Forschungsgemeinschaft through grants provided within TUM IGSSE (Biomat 05 PSN), the Gottfried–Wilhelm–Leibniz Program, the SFB863, and the Excellence Cluster CIPSM (Center for Integrated Protein Science Munich), the ERASynBio project ‘BioOrigami’, funded by Bundesministerium für Bildung und Forschung grant 031 A 458.
Extended data figures
This file contains sequences of all phagemids used in this work, of the chemically synthesized staples used for the pointer, as well as of all oligonucleotides used in the knock-out experiments.