Single-stranded DNA and RNA origami go live

Nanotechnologists are using DNA, the genetic material present in living organisms, as well as its multifunctional cousin RNA, as the raw material in efforts to build miniscule devices that could potentially function as drug delivery vehicles, tiny nanofactories for the production of pharmaceuticals and chemicals, or highly sensitive elements of electric and optical technologies.

Like genetic DNA (and RNA) in nature, these engineered nanotechnological devices are also made up of strands that are comprised of the four bases known in shorthand as A, C, T, and G. Regions within those strands can spontaneously fold and bind to each other via short complementary base sequences in which As from one sequence specifically bind to Ts from another sequence, and Cs to Gs. Researchers at the Wyss Institute of Biologically Inspired Engineering and elsewhere have used these features to design self-assembling nanostructures such as scaffolded DNA origami and DNA bricks with ever-growing sizes and complexities that are becoming useful for diverse applications. However, the translation of these structures into medical and industrial applications is still challenging, partially because these multi-stranded systems are prone to local defects due to missing stands. In addition, they self-assemble from hundreds to thousands of individual DNA sequences that each need to be verified and tested for high-precision applications, and whose expensive synthesis often produces undesired side products.

Now, a novel approach published in Science by a collaborative team of researchers from the Wyss Institute, Arizona State University, and Autodesk for the first time enables the design of complex single-stranded DNA and RNA origami that can autonomously fold into diverse, stable, user-defined structures. In contrast to the synthesis of multi-stranded nanostructures, these entirely new types of origami are folded from one single strand, which can be replicated in living cells, allowing their potential low-cost production at large scales and with high purities, opening entirely new opportunities for diverse applications such as drug delivery and nanofabrication.

Earlier generations of larger-sized origami are composed of a central scaffold strand whose folding and stability requires more than two hundred short staple strands that bridge distant parts of the scaffold and fix them in space. “In contrast to traditional scaffolded origamis, which are assembled from hundreds of components, our new approach allows us to reliably design and synthesize stable single-stranded and self-folding origami,” said Wyss Institute Core Faculty member and corresponding author Peng Yin, Ph.D. “Our fundamentally new approach relies on single-strand folding, rather than multi-component assembly, to produce large nanostructures. This, together with the ability to basically clone and multiply the single component strand in bacteria, presents a game-changing advance in DNA nanotechnology that greatly enhances single-stranded origami's potential for real-world applications.” Yin is also co-lead of the Wyss Institute's Molecular Robotics Initiative and Professor of Systems Biology at Harvard Medical School (HMS).

To first enable the production of single-stranded and stable DNA-based origami with distinct folding patterns, the team had to overcome several challenges. In a large DNA strand that goes through a complex folding process, many sequences need to accurately pair up with sequences that are far away from each other. If this process does not happen in an orderly and precise fashion, the strand gets tangled and forms unspecific knots along the way, rendering it useless. “To avoid this problem, we identified new design rules that we can use to cross DNA strands between different double-stranded regions and developed a web-based automated design tool that allows researchers to integrate many of these events into a folding path leading up to a large knot-free nanocomplex,” said Dongran Han, Ph.D., the study's first author and a Postdoctoral Fellow on Yin's team.

The largest DNA origami structures created previously were assembled by synthesizing all their constituent sequences individually in vitro and by mixing them together. As a key feature of the new design process, the single-strandedness of the DNA origami allowed the researchers to introduce DNA sequences stably into E. coli bacteria to inexpensively and accurately replicate them with every cell division. “This could greatly facilitate the development of single-stranded origami for high-precision nanotech like drug delivery vehicles, for example, as only a single easy-to-produce molecule needs to be validated and approved,” said Han.

Finally, the team also adapted single-stranded origami technology to RNA, which as a different nucleic acid material offers certain advantages including, for example, even higher production levels in bacteria, and usefulness for potential intra-cellular and therapeutic RNA applications. Translating the approach to RNA also scales up the size and complexity of synthetic RNA structures 10-fold compared to previous structures made from RNA.

Their proof-of-concept analysis also proved that protruding DNA loops can be precisely positioned and be used as handles for the attachment of functional proteins. In future developments, single-stranded origami could thus be potentially functionalized by attaching enzymes, fluorescent probes, metal particles, or drugs either to their surfaces or within cavities inside. This could effectively convert single-stranded origami into nanofactories, light-sensing and emitting optical devices, or drug delivery vehicles.

“This new advance by the Wyss Institute's Molecular Robotics Initiative transforms an exciting laboratory research methodology into a potentially transformative technology that can be manufactured at large scale by leveraging the biological machinery of living cells. This work opens a path by which DNA nanotechnology and origami approaches may be translated into products that meet real-world challenges,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children's Hospital, as well as Professor of Bioengineering at Harvard's John A. Paulson School of Engineering and Applied Sciences (SEAS).

“The results announced today establish DNA nanotechnology as a viable alternative approach for applications that have the potential to benefit all of us and the Nation as a whole,” said Jim Kurose, Assistant Director of the National Science Foundation's (NSF) Directorate for Computer and Information Science and Engineering (CISE). “We are delighted this work was supported by NSF's Expeditions in Computing program, which has, over the last decade funded large teams of researchers to pursue ambitious, fundamental research agendas that help define and shape the future of computer and information science and engineering, and impact our national competitiveness.”

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Besides Yin and Han, the study includes corresponding authors Hao Yan, Ph.D., and Fei Zhang, Ph.D., Director and Assistant Professor at the Biodesign Center for Molecular Design and Biomimetics at Arizona State University, Tempe, respectively, and Byoungkwon An, Ph.D., Principle Research Scientist at Autodesk Research, San Francisco; Shuoxing Jiang, Ph.D., Xiaodong Qi, and Yan Liu, Ph.D., Assistant Professor from the Biodesign Institute; Cameron Myhrvold, Ph.D., Bei Wang, and Mingjie Dai, Ph.D., past and present members of Yin's team at the Wyss Institute; and Maxwell Bates, who worked with An. The study was funded by the Office of Naval Research, the Army Research Office, the National Science Foundation's Expeditions in Computing program, and the Wyss Institute for Biologically Inspired Engineering.

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The Wyss Institute for Biologically Inspired Engineering at Harvard University uses Nature's design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world. Wyss researchers are developing innovative new engineering solutions for healthcare, energy, architecture, robotics, and manufacturing that are translated into commercial products and therapies through collaborations with clinical investigators, corporate alliances, and formation of new startups. The Wyss Institute creates transformative technological breakthroughs by engaging in high risk research, and crosses disciplinary and institutional barriers, working as an alliance that includes Harvard's Schools of Medicine, Engineering, Arts & Sciences and Design, and in partnership with Beth Israel Deaconess Medical Center, Brigham and Women's Hospital, Boston Children's Hospital, Dana-Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Boston University, Tufts University, Charité – Universitätsmedizin Berlin, University of Zurich and Massachusetts Institute of Technology.

Harvard Medical School has more than 11,000 faculty working in 10 academic departments located at the School's Boston campus or in hospital-based clinical departments at 15 Harvard-affiliated teaching hospitals and research institutes: Beth Israel Deaconess Medical Center, Boston Children's Hospital, Brigham and Women's Hospital, Cambridge Health Alliance, Dana-Farber Cancer Institute, Harvard Pilgrim Health Care Institute, Hebrew SeniorLife, Joslin Diabetes Center, Judge Baker Children's Center, Massachusetts Eye and Ear/Schepens Eye Research Institute, Massachusetts General Hospital, McLean Hospital, Mount Auburn Hospital, Spaulding Rehabilitation Network and VA Boston Healthcare System.