Biotechnological mass production of DNA origami

• Published in: Nature (London) Vol. 552; no. 7683; pp. 84 - 87
• Main Authors: Praetorius, Florian, Kick, Benjamin, Behler, Karl L., Honemann, Maximilian N., Weuster-Botz, Dirk, Dietz, Hendrik
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 07.12.2017; Nature Publishing Group
• Subjects: 101/28; 38/22; 45/29; 631/45/147; 631/61/350; 631/61/391; 639/925/926; 639/925/927; Bioreactors; Biotechnology; Chemical compounds; Deoxyribonucleic acid; DNA; DNA sequencing; Functional groups; Gating; Gene sequencing; Humanities and Social Sciences; letter; Mass production; Methods; Microscopy; Modularity; multidisciplinary; Nanorods; Nanotechnology; Nucleotide sequence; Oligonucleotides; Phages; Pharmacology; Production methods; Purification; Scaling; Science; Science and technology; Self-assembly; Single-stranded DNA; Strands; Zinc
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature24650

All necessary strands for DNA origami can be created in a single scalable process by using bacteriophages to generate single-stranded precursor DNA containing the target sequences interleaved with self-excising DNA enzymes. A mass of DNA origami DNA origami can readily be used to create micrometre-scale objects with nanometre-precise features using a very long single-stranded DNA 'scaffold' that is held in place by many short single-stranded DNA 'staples'. These objects could find many uses, but the cost of manufacturing them could be prohibitive for conducting research into their potential applications. Hendrik Dietz and colleagues now show that single strands of DNA of random length and sequence can be mass-produced at low cost. They used a litre-scale bioreactor to generate single-stranded precursor DNA strands that contain target strand sequences interspersed with self-excising DNA enzyme cassettes. This makes it possible to efficiently generate all of the single strands of DNA needed to assemble different target origami objects in one process, which should expand the scope of DNA nanotechnology in many areas of science and technology. Three related papers is this issue report further advances in DNA origami, and all four are summarized in a News & Views. DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 . These structures are customizable in that they can be site-specifically functionalized 13 or constructed to exhibit machine-like 14 , 15 or logic-gating behaviour 16 . 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 materials 3 , 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 production 23 ; the shorter staple strands are obtained through costly solid-phase synthesis 24 or enzymatic processes 25 . 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 Zn 2+ -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.