Researchers Use Single-Stranded DNA, RNA to Create Self-Assembling Nanostructures

Nanotechnologists are making use of DNA, the genetic material that is present in living organisms, as well as its multifunctional counterpart RNA, as the raw material in attempts to design miniscule devices that could potentially function as drug delivery vehicles, miniature nanofactories for the production of chemicals and pharmaceuticals, or extremely sensitive elements of optical and electric technologies.

Similar to genetic DNA (and RNA) in nature, these engineered nanotechnological devices are also composed of strands that comprise the four bases known in shorthand as A, C, T, and G. Regions within those strands can naturally fold and bind to each other via short complementary base sequences in which As from one sequence precisely 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 engineer self-assembling nanostructures such as scaffolded DNA origami and DNA bricks with ever-increasing sizes and complexities that are becoming beneficial for varied applications. However, the translation of these structures into industrial and medical applications is still challenging, partly because these multi-stranded systems are susceptible to local defects as a result of missing stands. Furthermore, they self-assemble from hundreds to thousands of separate DNA sequences that each need to be confirmed and tested for high-precision applications, and whose costly synthesis repeatedly produces undesired by-products.

Currently, an innovative approach published in the Science journal by a collaborative team of researchers from the Wyss Institute, Arizona State University, and Autodesk for the first time enables the design of multifaceted 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 completely 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 whole new opportunities for various applications such as nanofabrication and drug delivery.

Previous generations of larger-sized origami are made up of a principal scaffold strand whose folding and stability requires over 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, 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.

Peng Yin, Ph.D, Wyss Institute Core Faculty member and corresponding author.

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 manufacture of single-stranded and stable DNA-based origami with diverse folding patterns, the team had to overcome numerous challenges. In a large DNA strand that goes through an intricate folding process, many sequences have to accurately pair up with sequences that are not close to each other. If this process does not happen in a methodical and precise manner, the strand gets twisted and forms undefined knots along the way, rendering it unusable. “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 developed earlier were put together by synthesizing all their constituent sequences separately in vitro and by combining them together. As a main feature of the new design process, the single-strandedness of the DNA origami allowed the researchers to add DNA sequences stably into E. coli bacteria to economically and accurately reproduce 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 provides specific benefits including, for instance, even higher production levels in bacteria, and practicality for potential intra-cellular and therapeutic RNA applications. Translating the method to RNA also scales up the complexity and size of artificial RNA structures 10-fold compared to earlier structures made from RNA.

Their proof-of-concept analysis also proved that protruding DNA loops can be exactly placed and be used as handles for the attachment of functional proteins. In forthcoming developments, single-stranded origami could therefore be potentially functionalized by attaching enzymes, metal particles, fluorescent probes, or drugs either to their surfaces or within cavities. This could efficiently 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, 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.

Jim Kurose, Assistant Director of the National Science Foundation’s (NSF) Directorate for Computer and Information Science and Engineering (CISE).

Besides Yin and Han, the research 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 research received funding from 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.

In this animation, a long single-strand of DNA is self-folding into a highly programmable and complex origami nanostructure significantly larger than those created in previous attempts. Credit: Molgraphics