Angewandte Reviews Chemie International Edition:DOI:10.1002/anie.201707976 DNA Nanotechnology German Edition:DOI:10.1002/ange.201707976 From Designing the Molecules of Life to Designing Life:Future Applications Derived from Advances in DNATechnologies Richie E. Kohman+,Aditya M. Kunjapur+,Eriona Hysolli+,YuWang+,and George M. Church* Keywords: DNA nanotechnology · DNA synthesis · genome engineering · genome recoding · synthetic biology Angewandte Chemie Angew.Chem. Int.Ed. 2018, 57,4313 –4328 2018 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim www.angewandte.org 4313 GDCh Angewandte Reviews ,... .....- ....... Chemie """"""""' Since the elucidation of its structure, DNA has been at the forefront of From the Contents biological research. In the past half century, an explosion of DNA­ based technology development has occurred with the most rapid 1. Introduction 43 74 advances being made for DNA sequencing. In parallel, dramatic 2. How Advancements in DNA improvements have also been made in the synthesis and editing of Synthesis Will Effect DNA from the oligonucleotide to the genome scale. In this Review, we Nanotechnology 43 74 will summarize four different subfields relating to DNA technologies following this trajectory of smaller to larger scale. We begin by talking 3· The Future of Microbial Genome Recoding and about building materials out of DNA which in turn can act as delivery Synthesis 43 76 vehicles in vivo. We then discuss how altering microbial genomes can lead to novel methods of production for industrial biologics. Next, we 4· Writing Genomes to Explore talk about the future of writing whole genomes as a method ofstudying Evolution 4320 evolution. Lastly, we highlight the ways in which barcoding biological 5· The Use of DNA as Information systems will allow for their three-dimensional analysis in a highly Carriers for In Situ Omics multiplexed fashion. Analysis 4322 6. Summary and Outlook 4324 1. Introduction Technology to read, write, and edit DNA is central to all fields of current biological investigation. For decades, con­ tinuing effort has been made to improve upon these three DNA-based technologies. The most rapid advances have been decades, numerous methods for building nanostructures out made in the ability to sequence DNA, as the cost per base had of DNA have been demonstrated. As expected, progress in dropped by one million-fold in three decades. This massive DNA nanotechnology is closely influenced by DNA synthesis technological improvement has truly changed the way biology technology. Here we entertain the question of how this field is investigated with sequencing being a commonplace tool. In would be effected if longer and cheaper DNA was readily parallel, technologies to write and edit DNA have seen available. Although relevant for all DNA nanotechnology,[2J similar advances in recent years. Due to improvements in here we indulge upon how this technological leap will instrumentation, the cost of DNA synthesis continues to specifically effect applications of DNA origami-based nano­ decrease at rates faster than predicted, while our ability to technology for in vivo delivery. edit genomes has been dramatically enhanced due to key DNA origami is a technique developed in 2006 that discoveries in the field of endonucleases. involves the self-assembly of a large strand of DNA (the The technologies enabled by these advances are numer­ scaffold) with many small pieces of DNA (the staples) to ous, and continue to flourish. Due to the decrease in the cost generate well-defined nanostructures (Figure 1 A)Yl In a rel­ of DNA synthesis and our understanding of the functional atively short period of time, numerous advances have been properties of various genetic elements, the behavior of entire made to the original methodology[4J such as methods to create cells can now be programmed to carry out specific operations. three-dimensional structures with and without curvature[sJ Additionally, DNA can now be used in a non-canonical using the assembly of two-dimensional sheets,[6J packing of fashion to influence and study biological systems through the multiple sheets at a variety of densities,Pl or using a wireframe use of multiplexed DNA tags or through the construction of approach_[sJ Design software was created to drastically DNA-based materials. In this Review, we will highlight recent advances in several DNA-based technologies and discuss what their future ['''] Dr. A. M. Kunjapur,l+l Dr. E. Hysolli,l+l Y. Wang,l+l Prof. G. M. Church applications will be. We will follow the trajectory of increasing Department of Genetics, Harvard Medical School scale and complexity, beginning with engineering DNA Boston, MA 02115 (USA) nanomaterials and continuing towards engineering genomes and organisms. Dr. R. E. Kohman,l+l Y. Wang, I+J Prof. G. M. Church Wyss Institute for Biologically Inspired Engineering Harvard University Boston, MA 02115 (USA) 2. How Advancements in DNA Synthesis Will Effect [+] These authors contributed equally to this work. Nanotechnology G) The ORCID identification number(s) for the author(s) of this article can be found under https:f jdoi.org/1 0.1002fanie.201707976. The availability of synthetic DNA has allowed researchers M This article is part of the Special Issue to commemorate the 350th to utilize it as a nanofabrication substrate rather than just the anniversary of Merck KGaA, Darmstadt, Germany. More articles can substrate for biological information storage. [!J Over several be found at http :f jdoi.wiley.comf1 0.1 002fanie.v57.16. 4314 Wiley Online Library © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chern. Int. Ed. 2018, 57, 4313-4328 Angewandte Reviews Chemie simplify the design process,[9] and techniques were developed ability to undergo conformational changes when exposed to that improved both folding[10] and purification[11] of the intrinsic,such as endogenous intracellular biomolecules or nanostructures.Itisnow possible to design, create,and pH changes,orextrinsic triggers,such as exposure to characterize anovel DNAnanostructure within afew chemicals[13] or light.[14] Taken together,these properties weeks.[12] make DNAnanostructures optimal candidates to address DNAorigami nanostructures have avariety of unique many of the unmet challenges in bio-nanotechnology,such as properties that make them ideal for biological applications the in vivo targeting and delivery of therapeutics and such as drug delivery,enzymatic nanoreactors,and bioanal- diagnostics.Progress has already been made toward this ysis:[4] 1) Thedesign space is large,asitispossible to create end. Chemotherapeutic intercalators have been bound to uniform structures of arbitrary two- and three-dimensional packed origami helices and delivered to cancer cells after shapes;2)nanostructure creation is relatively straightforward being internalized (Figure 1B).[15] Small molecules neuro- with most designs folding successfully in one thermal anneal- transmitters as well as full proteins have been released from ing step over the course of 12 to 48 hours;3)DNA origami the cavities of DNAnanostructures with light-triggered nanostructures are highly addressable as functional handles uncaging (Figure 1C).[16] Also,wehave recently designed can be positioned at precise locations along their surfaces; cancer-killing nano-robots which underwent structural con- and 4) structures can possess dynamic properties such as the formations in the presence of environmental cues to expose cell-killing antibodies previously hidden within their interior Richie E. Kohman is aresearch scientist and (Figure 1D).[17] Lastly,toaddress stability in in vivo environ- member of the Advanced Technology Team ments,surface modifications using lipid bilayers (Fig- at the Wyss Institute for Biologically Inspired ure 1E)[18] and hydrophilic polymers[19] have been developed. EngineeringatHarvard University in the Despite these advances,several issues need to be laboratory of Professor George Church. His addressed before DNAorigami nanostructures can effec- reseach interests lie in nucleic acid chemistry, nanotechnology,and neurotechnology.He tively scale for many biological applications.The cost of the obtained his B.S. in Chemistry from Santa synthetic oligonucleotides used for the staple DNAisstill Clara University and his Ph.D. in Chemistry substantial compared to synthetic materials and must be from the University of Illinois Urbana-Cham- lowered to be able to create affordable therapies and paign. He then pursued postdoctoral diagnostics.This will most likely require substantial improve- research in the Department of Biomedical ments in DNAsynthesis technology,such as using biosyn- EngineeringatBoston University and as an thetic methods as opposed to purely chemical ones.[20] An affliate in the Media Lab at the Massachusetts Institute of Technology. Prior to working at the Wyss Institute, he served as Group Leader at additional issue is that the scaffold DNAexceeds the length ExpansionTechnologies, Inc. that can be chemically synthesized and thus prevents Aditya M. Kunjapur is aPostdoctoral Yu Wang is aPh.D. student in the laboratory Research Fellow in the laboratory of Profes- of Professor George Church in the Depart- sor George Church in the Department of ment of Genetics at Harvard Medical Genetics at Harvard Medical School. He is School and Wyss Institute for Biologically focused on developing high-throughput Inspired EngineeringatHarvard University. methods to selectively introduce novel func- His research interestsare focused on highly tional groups