Time for DNA Manufacturing reduced from weeks to minutes and process for validation of the manufactured DNA structures enabled

Eurekalert – Two major barriers to the advancement of DNA nanotechnology beyond the research lab have been knocked down. This emerging technology employs DNA as a programmable building material for self-assembled, nanometer-scale structures. Many practical applications have been envisioned, and researchers recently demonstrated a synthetic membrane channel made from DNA. Until now, however, design processes were hobbled by a lack of structural feedback. Assembly was slow and often of poor quality. Now researchers led by Prof. Hendrik Dietz of the Technische Universitaet Muenchen (TUM) have removed these obstacles.

One barrier holding the field back was an unproven assumption. Researchers were able to design a wide variety of discrete objects and specify exactly how DNA strands should zip together and fold into the desired shapes. They could show that the resulting nanostructures closely matched the designs. Still lacking, though, was the validation of the assumed subnanometer-scale precise positional control. This has been confirmed for the first time through analysis of a test object designed specifically for the purpose. A technical breakthrough based on advances in fundamental understanding, this demonstration has provided a crucial reality check for DNA nanotechnology.

In a separate set of experiments, the researchers discovered that the time it takes to make a batch of complex DNA-based objects can be cut from a week to a matter of minutes, and that the yield can be nearly 100%. They showed for the first time that at a constant temperature, hundreds of DNA strands can fold cooperatively to form an object — correctly, as designed — within minutes. Surprisingly, they say, the process is similar to protein folding, despite significant chemical and structural differences. “Seeing this combination of rapid folding and high yield,” Dietz says, “we have a stronger sense than ever that DNA nanotechnology could lead to a new kind of manufacturing, with a commercial, even industrial future.” And there are immediate benefits, he adds: “Now we don’t have to wait a week for feedback on an experimental design, and multi-step assembly processes have suddenly become so much more practical.”

Caption: This 3-D print shows a DNA-based structure designed to test a critical assumption — that such objects could be realized, as designed, with subnanometer precision. This object is a relatively large, three-dimensional DNA-based structure, asymmetrical to help determine the orientation, and incorporating distinctive design motifs. Subnanometer-resolution imaging with low-temperature electron microscopy enabled researchers to map the object — which comprises more than 460,000 atoms — with subnanometer-scale detail. Credit: Dietz Lab, TU Muenchen

Three research papers detail the work

Science – Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature

ABSTRACT – We demonstrate that, at constant temperature, hundreds of DNA strands can cooperatively fold a long template DNA strand within minutes into complex nanoscale objects. Folding occurred out of equilibrium along nucleation-driven pathways at temperatures that could be influenced by the choice of sequences, strand lengths, and chain topology. Unfolding occurred in apparent equilibrium at higher temperatures than those for folding. Folding at optimized constant temperatures enabled the rapid production of three-dimensional DNA objects with yields that approached 100%. The results point to similarities with protein folding in spite of chemical and structural differences. The possibility for rapid and high-yield assembly will enable DNA nanotechnology for practical applications.

The usual self-assembly process is often described as a “one-pot reaction”: Strands of DNA that will serve as the template, instructions, and building material for a designed object are placed together at a relatively high temperature where they will remain separate; the temperature is gradually lowered, and somewhere along the line the DNA strands zip together to form the desired structures.

Observing this process in unprecedented detail, the TUM researchers discovered that all of the action takes place within a specific and relatively narrow temperature range, which differs depending on the design of the object. One practical implication is that, once the optimal temperature for a given design has been determined, DNA self-assembly – nanomanufacturing, in essence – could be accomplished through fast processes at constant temperatures. Following up on this lead, the researchers found that they could “mass-produce” objects made from hundreds of DNA strands within minutes instead of days, with almost no defective objects or by-products in the resulting batch.

“Besides telling us that complex DNA objects are manufacturable,” Dietz says, “these results suggest something we hardly dared to imagine before – that it might be possible to assemble DNA nanodevices in a cell culture or even within a living cell.”

From the viewpoint of fundamental biology, the most intriguing result of these experiments may be the discovery that DNA folding resembles protein folding more closely than anticipated. Chemically and structurally, the two families of biomolecules are quite different. But the researchers observed clearly defined “cooperative” steps in the folding of complex DNA objects, no different in principle from mechanisms at work in protein folding. They speculate that further experiments with self-assembly of designed DNA objects could help to unravel the mysteries of protein folding, which is more complex and less accessible to direct study.

47 pages of supplemental material

PNAS – Cryo-EM structure of a 3D DNA-origami object

Abstract – A key goal for nanotechnology is to design synthetic objects that may ultimately achieve functionalities known today only from natural macromolecular complexes. Molecular self-assembly with DNA has shown potential for creating user-defined 3D scaffolds, but the level of attainable positional accuracy has been unclear. Here we report the cryo-EM structure and a full pseudoatomic model of a discrete DNA object that is almost twice the size of a prokaryotic ribosome. The structure provides a variety of stable, previously undescribed DNA topologies for future use in nanotechnology and experimental evidence that discrete 3D DNA scaffolds allow the positioning of user-defined structural motifs with an accuracy that is similar to that observed in natural macromolecules. Thereby, our results indicate an attractive route to fabricate nanoscale devices that achieve complex functionalities by DNA-templated design steered by structural feedback

8 pages of supplemental material for the PNAS research

Atomically precise control

To test the assumption that discrete DNA objects could be assembled as designed with subnanometer precision, TUM biophysicists collaborated with scientists at the MRC Laboratory of Molecular Biology in Cambridge, UK. They produced a relatively large, three-dimensional DNA-based structure, asymmetrical to help determine the orientation, and incorporating distinctive design motifs.

Subnanometer-resolution imaging with low-temperature electron microscopy enabled the researchers to map the object — which comprises more than 460,000 atoms — with subnanometer-scale detail. Because the object incorporates, in effect, a whole library of different design elements, it will also serve as a resource for further study. The results, reported in Proceedings of the National Academy of Sciences, not only demonstrate atomically precise assembly, but also show that such structures, formerly thought to be jelly-like and flexible, are rigid enough to be probed by electron microscopy.

Science – Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures

ABSTRACT – We created nanometer-scale transmembrane channels in lipid bilayers by means of self-assembled DNA-based nanostructures. Scaffolded DNA origami was used to create a stem that penetrated and spanned a lipid membrane, as well as a barrel-shaped cap that adhered to the membrane, in part via 26 cholesterol moieties. In single-channel electrophysiological measurements, we found similarities to the response of natural ion channels, such as conductances on the order of 1 nanosiemens and channel gating. More pronounced gating was seen for mutations in which a single DNA strand of the stem protruded into the channel. Single-molecule translocation experiments show that the synthetic channels can be used to discriminate single DNA molecules.

43 pages of Supplementary Materials for Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures

Sources for this article

Eurekalert announcement – Reality check for DNA nanotechnology. – from Technische Universitaet Muenchen

Original publications:

Xiao-chen Bai, Thomas G. Martin, Sjors H. W. Scheres, Hendrik Dietz. Cryo-EM structure of a 3D DNA-origami object. Proceedings of the National Academy of Sciences of the USA, Dec. 4, 2012, 109 (49) 20012-20017; on-line in PNAS Early Edition, Nov. 19, 2012. DOI: 10.1073/pnas.1215713109

Jean-Philippe J. Sobczak, Thomas G. Martin, Thomas Gerling, Hendrik Dietz. Rapid folding of DNA into nanoscale shapes at constant temperature. Science, vol. 338, issue 6113, pp. 1458-1461. DOI: 10.1126/science.1229919

See also: Martin Langecker, Vera Arnaut, Thomas G. Martin, Jonathan List, Stephan Renner, Michael Mayer, Hendrik Dietz, and Friedrich C. Simmel. Synthetic lipid membrane channels formed by designed DNA nanostructures. Science, vol. 338, issue 6109, pp. 932-936. DOI: 10.1126/science.1225624

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