Biotechnological mass production of DNA origami

Abstract

DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behaviour. 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, 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; the shorter staple strands are obtained through costly solid-phase synthesis or enzymatic processes. 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-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.

DNA self-assembly scaled up

“…Praetorius et al 5 (who belong to the same research group as Wagenbauer and colleagues; page 84) report biotechnology that should greatly reduce the cost of the hundreds of staple strands that are usually used to make DNA origami. They use viruses known as bacteriophages to produce single-stranded precursor DNA that contains hundreds of staple-strand sequences.…”

“…Scientists have long aspired to construct artificial objects using self-assembly to reach the dimensions and complexity of cells or organelles, with the aim of building synthetic cellular machines for research, engineering and medical applications. Four papers [2][3][4][5] in this issue address this goal by reporting methods for scaling up the sizes and production of self-assembling, designer nanostructures made from DNA.…”

Beyond the smiley face: applications of structural DNA nanotechnology

“…Furthermore, recent development of mass-produced scaffold molecules via bacteriophages have shown promise in developing cost-effective DNA origami structures of any length [16]. Within a shaker-flask culture, bacteriophages produce a ‘precursor’ DNA which contains target strand sequences and self-excising Zn 2+ dependent ‘cassettes’ containing DNA-cleaving DNA enzymes, to produce single strands that can be modifiable within any existing DNA origami design framework [16].…”

“…Within a shaker-flask culture, bacteriophages produce a ‘precursor’ DNA which contains target strand sequences and self-excising Zn 2+ dependent ‘cassettes’ containing DNA-cleaving DNA enzymes, to produce single strands that can be modifiable within any existing DNA origami design framework [16]. …”

Bigger and cheaper

“…Now a series of papers showcasing micrometresized two-dimensional DNA arrays and gigadalton three-dimensional origami structures might move the field a little bit closer to this goal, proving that bigger and more complex architectures can be built with high yields and low error rates [3][4][5] . Notably, a smart one-pot biotechnological approach published alongside these papers also offers a pragmatic route to cut DNA origami production costs 6 . In this strategy, bacteriophages are programmed to amplify a precisely engineered, self-cleaving DNA template that generates both the scaffold and the staple sequences needed for 'in-phage' self-assembly.…”