DNA, the medium of life, is so deeply associated with the biochemical world that considering its nonbiological applications might appear far-fetched. Nevertheless, for researchers in the 1980s and 1990s operating in the recently established field of DNA nanotechnology, it was more than a flight of fancy. Nadrian “Ned” Seeman, the daddy of DNA nanotechnology and previously a biochemist at New York University, very first proposed the concept that DNA is not only a hereditary product but also a building material in his critical 1982 Journal of Theoretical Biology paper.1 A crystallographer by training, Seeman had a hard time to crystalize proteins. He wanted to develop DNA cages that were strong enough to hold the protein in location long enough to take an excellent photo. En path to tackling this problem, Seeman produced the field of DNA nanotechnology. “Ned had such a huge body of literature that hes sort of everybodys go-to inspiration source,” stated Erik Winfree, a computer system researcher and bioengineer at the California Institute of Technology (Caltech). [Rothemunds] presentation was so elegant and comprehensive that it actually opened individualss eyes to how effective the concept was. — Erik Winfree, California Institute of TechnologyIn the years that followed, grand ideas transformed into even grander demonstrations as researchers repurposed nucleic acids to store nonbiological info, such as all 154 of William Shakespeares sonnets, and construct tiny robotics for drug shipment.2,3 DNA computersIn the early 1990s as an undergraduate student at Caltech, Paul Rothemund, now a computer system scientist there, took a computer technology class that introduced him to the potential of DNA. The professor discussed a concept from Charles Bennett, then a physicist at IBM, that a computer system built from DNA might mimic a Turing maker in which theoretical enzymes might check out the details saved in DNA and use that information to modify DNA bases.4 “He stated, you understand, sooner or later, someone who knows something about computer system science and biology or chemistry will come up with a way to calculate utilizing DNA. At that minute, I stated, Well, maybe I can do that,” recalled Rothemund. He didnt have to wait long. Rothemund had the chance to even more explore the concept of constructing a molecular computer system for a task in another class. There, Rothemund fulfilled Winfree, then a graduate trainee, and presented him to Seemans work. Erik Winfree (left), Bernard Yurke (middle), and Paul Rothemund (right) explore methods to expand the use of DNA.Erik WinfreeAs Rothemund neared the end of his research studies, he wanted to continue working on the problem of building DNA computers, so he went shopping the project around. Computer technology teachers felt ill-equipped to recommend on the life science aspects of the task, so he turned to life researchers. “The biology teachers at Caltech and in other places told me that I was crazy, and they had no concept what I was discussing,” stated Rothemund. He hit a dead end, and following graduation in 1994, he signed up with a geobiology laboratory as a professional. Later on that year, Leonard Adleman, a computer scientist at the University of Southern California (USC), published an influential paper in Science in which he used DNA to compute an algorithm.5 “I at the same time felt sort of scooped and verified that there was something to the concept of encoding details in DNA particles and doing computing,” said Rothemund. Winfree remembered Rothemunds class job on a comparable topic and hunted him down to see if he wished to participate in the inaugural one day workshop on DNA-based computers that would be held at Princeton University in the spring of 1995. They hunted up the cash to go to the conference. Winfrees talk touched on Seemans deal with the self-assembly of DNA structures: the spontaneous organization of molecules due to appealing forces.After going back to his seat, Winfree felt Seeman pull on his arm. Later on that evening, Adleman, Seeman, Rothemund, and Winfree gathered over a white and red checkered tablecloth in a pizza parlor and assessed the days events. “That sort of seeded the next 25 years of my life,” said Rothemund. He signed up with Adlemans laboratory at USC later on that year as a college student, while Winfree ventured to the east coast to work together with Seeman at New York University on the self-assembly of DNA crystals.6 Folding DNADNA computing captured the creativities of lots of entering the nascent field of DNA nanotechnology by demonstrating a nonbiological application for nucleic acids. By the turn of the century, lots of scientists shifted their focus. “Many in the field convinced ourselves that although this was intellectually promoting, this was not going to complete with electronic computer systems,” stated Winfree. Rothemund focused his efforts on structure nanostructures using DNA, or programmable approaches for DNA assembly. Specifically, he considered how self-assembly processes might be dealt with as algorithms and studied using computer science tools. In 1993, Seeman detailed the construction of complicated nanostructures via the self-assembly of particles.7 He had an interest in determining how to develop molecules that self-assemble to form parallel DNA helices where hairs cross over and enter into another double helical line, therefore sewing together the helices. Over the next years, this inspired others in the field to innovate, increasing the number of crossovers and helices.Rothemund and Winfrees courses crossed again in 2001. Winfree went back to Caltech as a teacher, and Rothemund joined his lab as a postdoctoral researcher, where they continued their work on algorithmic self-assembly of DNA.8 DNA is a flexible building block, but Rothemund described DNA as optically, biochemically, and digitally dead compared to other molecules and products like quantum dots, carbon nanotubes, or antibodies.”But what it can do is you can use the details in DNA series to construct structures, and after that you can use that structure to arrange those other things,” stated Rothemund. Since the available methods for producing shapes out of DNA were laborious and time consuming, Rothemund set out to establish a much easier technique. “And truly, that was the idea for DNA origami,” he stated. For the very first DNA origami experiments, Rothemund developed one-third of a square. By adding only one-third of the required staple hairs, just one-third of the square folded, leading to rectangle-shaped shapes caught using atomic force microscopy. The “ss” label denotes unfolded single-stranded scaffold; “s, m” represents a steady monomer; and “u, m” denotes an unstable monomer. The scale bar is 100 nm.Paul RothemundRothemunds DNA origami consisted of two main components: a long, single-stranded piece of bacteriophage DNA, which serves as the scaffold material, and a bunch of shorter strands of oligonucleotides, or staple hairs, that fix the structure in place.9 He fed his design into a computer system program that utilized concepts of Watson-Crick base pairing to figure out which sequences were required to advise the scaffold strand to fold into the desired shape or pattern. In his one-pot method, Rothemund blended the scaffold hair with the personalized staple strands and waited patiently as molecular self-assembly took shape. To validate the structures, he used atomic force microscopy.At the time, Winfree gave his laboratory members the flexibility to separately explore their interests. “There was a duration in 2005 when I didnt see [Rothemund] around really much,” stated Winfree. Eventually, Rothemund reappeared with something to share. “He showed me his results on the DNA origami, and to tell you the fact, my very first response was Blech! Wheres the algorithm? Wheres computer science?” Uninterested in working together on the task, Winfree recommended that Rothemund release it himself, therefore he did. Winfree eventually happened to DNA origami. “Its wonderful. Its changed the field,” stated Winfree.” [Rothemunds] presentation was so classy and comprehensive that it truly opened peoples eyes to how effective the concept was.”DNA origamis spiritual successorsDNA origami isnt the only technique for DNA assembly, but its relatively simple and robust.10 “Its the capability to produce a geometrically structured testbed for your explore each particle in the right location, and now, hundreds to countless particles,” stated Winfree. “That was extraordinary. That opened a capability to do experiments in all sorts of fields that people previously could not do.” For Winfree, that was putting brief DNA series in the proper order to set off self-assembly. For others, it was putting enzymes in the best order to produce a waterfall of enzyme reactions or quantum dots in a particular company to control optics. “One of the important things that DNA origami has actually had the ability to do because that paper was released is not something thats commonly used in everybodys cellular phone or something like that, however its a research tool for other things,” said Rothemund. He kept in mind that these customized instruments for biology allow scientists to begin asking concerns about proteins or other biomolecules and even translate these concepts into therapies and molecular diagnostics.11,12 I saw his talk, and my jaw dropped due to the fact that these images that he produced– no one had seen anything like this. — William Shih, Harvard Medical SchoolWilliam Shih, a biochemist at Harvard Medical School, employs principles of DNA origami in his quest to develop nanoscale objects, including molecular robots. In 2005, at a conference in Albany, Shih learned what Rothemund had actually depended on. “I saw his talk, and my jaw dropped since these images that he produced– nobody had seen anything like this,” said Shih. He remembered an especially unforgettable image of DNA origami with “Ned” patterned on top in homage to Seeman. Still enthralled by Rothemunds creations, Shih went back to his laboratory, ditched what he was doing, and rotated to Rothemund-style DNA origami. “To me, whats most unique about DNA origami is that you have an excess of structure blocks that do definitely nothing except when they see a copy of this master controller scaffold hair,” stated Shih. Ten copies of the scaffold strand produce 10 DNA origami structures if there suffice staple hairs. Around the very same time that Rothemund tinkered with DNA at Caltech, Shih worked as a postdoctoral researcher down the roadway at the Scripps Research Institute. In 2004, he published a paper in Nature showing the building and construction of a nanoscale octahedron.13 However, Shihs DNA folding approach needed the assembly of a substantial number of short, cloneable DNA structures. Shih kept in mind that without a master scaffold hair, the building and construction is more difficult to manage. “Its sort of like herding felines,” stated Shih. Just recently, Shihs research group has been hectic developing what he described as the spiritual followers of DNA origami. The scale of DNA origami structures is limited by the length of the scaffold hairs, which is typically on the order of 10,000 nucleotides. Constructing anything bigger than that requires passing up the leading scaffold hair. To resolve this, Shih and his group just recently established crisscross DNA origami where a controller particle on the scale of a single DNA origami directs the building of a larger 1,000 DNA origami structure.14 “Theyre more like well-trained pets than cats,” stated Shih.Shih hopes that these advances will assist in the construction of larger nanorobots with the size and complexity of a mammalian or bacterial cell. He views this as a complementary innovation development to the wider field of synthetic biology where researchers customize the genomes of living cells. “But theyre still living cells,” said Shih. Theyre still surrounded by a membrane; they still have metabolic process; and they still do DNA replication. “Its essential, technically, to have alternate schemes that maybe dont have a membrane, that are not beholden to the normal process of DNA duplication and translation, that perhaps can be deployed in environments that are hostile to living cells,” stated Shih. ReferencesSeeman NC. Nucleic acid junctions and lattices. J Theor Biol. 1982; 99( 2 ):237 -247. Goldman N, et al. Towards practical, high-capacity, low-maintenance details storage in manufactured DNA. Nature. 2013; 494:77 -80. Douglas SM, et al. A logic-gated nanorobot for targeted transport of molecular payloads. Science. 2012; 335( 6070 ):831 -834. Bennett CH. Sensible reversibility of calculation. IBM J Res Develop. 1973; 17( 6 ):525 -532. Adleman LM. Molecular calculation of options to combinatorial issues. Science. 1994; 266( 5187 ):1021 -1024. Winfree E, et al. Design and self-assembly of two-dimensional DNA crystals. Nature. 1998; 394( 6693 ):539 -544. Fu TJ, Seeman NC. DNA double-crossover particles. Biochemistry. 1993; 32( 13 ):3211 -3220. Rothemund PWK, et al. Algorithmic self-assembly of DNA Sierpinski triangles. PLoS Biol. 2004; 2( 12 ): e424.Rothemund PWK. Folding DNA to develop nanoscale shapes and patterns. Nature. 2006; 440:297 -302. LaBean TH. Reminiscences from the trenches: The early years of DNA nanotech. In: Jonoska N, Winfree E, eds. Visions of DNA Nanotechnology at 40 for the Next 40. Natural Computing Series. Springer, Singapore; 2023:55 -67. Andersen ES, et al. Self-assembly of a nanoscale DNA box with a controllable lid. Nature. 2009; 459:73 -76. Ochmann SE, et al. Optical nanoantenna for single molecule-based detection of Zika infection nucleic acids without molecular multiplication. Anal Chem. 2017; 89( 23 ):13000 -13007. Shih WM, et al. A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature. 2004; 427:618 -621. Wintersinger CM, et al. Multi-micron crisscross structures grown from DNA-origami slats. Nat Nanotechnol. 2023; 18( 3 ):281 -289.
Rothemund focused his efforts on building nanostructures using DNA, or programmable methods for DNA assembly. Winfree returned to Caltech as a professor, and Rothemund joined his lab as a postdoctoral researcher, where they continued their work on algorithmic self-assembly of DNA.8 DNA is a versatile building block, however Rothemund described DNA as optically, biochemically, and electronically dead compared to other molecules and materials like quantum dots, carbon nanotubes, or antibodies. The scale bar is 100 nm.Paul RothemundRothemunds DNA origami consisted of two primary parts: a long, single-stranded piece of bacteriophage DNA, which serves as the scaffold product, and a bunch of shorter strands of oligonucleotides, or staple strands, that fix the structure in place.9 He fed his design into a computer system program that used concepts of Watson-Crick base pairing to figure out which sequences were required to instruct the scaffold hair to fold into the preferred shape or pattern.”DNA origamis spiritual successorsDNA origami isnt the only method for DNA assembly, however its relatively simple and robust.10 “Its the capability to create a geometrically structured testbed for your experiment with each particle in the best location, and now, hundreds to thousands of molecules,” said Winfree. To address this, Shih and his group recently developed crisscross DNA origami where a controller molecule on the scale of a single DNA origami directs the building of a bigger 1,000 DNA origami structure.14 “Theyre more like well-trained canines than cats,” stated Shih.Shih hopes that these advances will facilitate the building and construction of larger nanorobots with the size and intricacy of a bacterial or mammalian cell.