Experience of Hands-on training program on DNA Nanomachine at IUPAB2024

The concept of the International Union for Pure and Applied Biophysics 2024 (IUPAB2024) is “Rocking Out Biophysics,” which means we will shake up biophysics at this conference and create new biophysics together. This congress provided more than 30 sessions that featured invited talks by leading scientists in their fields. In addition, this congress prepared Hands-on training programs for young researchers to experience front-line biophysics research in Japan. I obtained the opportunity to attend the Hands-on Training program D: DNA Nanomachine tutorial. Based on the concepts of this conference, I would like to share my experience from participating in the Hands-on Training Program with thanks to everyone involved.

The Hands-on Training program D: DNA Nanomachine tutorial was held at Kansai University supported by Grant-in-Aid for Transformative Research Areas (A) “Molecular cybernetics.” The program offered a hands-on program for young researchers interested in molecular robotics to learn the basics of designing and evaluating DNA nanomachines in a short time. The program is mainly divided into four parts: a lecture, a hands-on part, a networking part, and a session at the congress. I will briefly introduce and share my impressions of each part.

At first, the history of DNA nanomachines was discussed. In 1982, N. C. Seeman (New York University) proposed a method for creating lattice structures by self-assembling four-armed DNA structures known as Holliday Junctions [1]. Subsequently, three-dimensional structures such as cubes were also constructed through the self-assembly of DNA. In 1998, using a double crossover structure, which involves linking two double-stranded DNAs at two points, Seeman’s research group successfully created two-dimensional assemblies of DNA structures observable by atomic force microscopy (AFM) [2]. This breakthrough led to the birth of the field of “DNA nanotechnology,” which focuses on constructing structures through the self-assembly of DNA. Then, in 2006, Rothermund (California Institute of Technology) developed the design and creation strategy of the DNA nanostructure, which is approximately 100 nm in size and is called DNA origami [3]. By adding short complementary DNA strands (staple DNA) to a circular single-stranded DNA template (about 7,000 bases long, scaffold DNA) and then heating and slowly cooling (annealing) the mixture, DNA can be molecularly assembled into the pre-designed nanostructures. For example, the smiley face DNA nanostructure, the most famous DNA origami (cover image of Nature), was successfully demonstrated. Subsequently, methods for constructing three-dimensional DNA origami structures were established by adding vertical geometric bonds between double-stranded DNA molecules to the planar structures. The software “cadnano” was also developed for designing DNA origami [4].

At hands-on training, participants learn how to design DNA origami through cadnano. A brief procedure for the software was introduced. Initially, we created double-stranded DNA. In the Slice window of the software, the number of DNA strands was selected. Then, in the Path window, the number of bases was adjusted. In the software, DNA is represented by arrows (Fig. 1a). Next, the structure of DNA with crossovers was designed. Two double-stranded DNAs were prepared. The crossover was formed using the L-shaped button displayed between the two double-stranded DNAs (Fig. 1b). Finally, we designed a trapezoidal DNA nanostructure. In the Slice window, multiple handles were prepared, and a circular DNA was formed. The staple DNAs were automatically formed by the “AutoStaple” button (Fig. 1c). Then, the positions of the crossovers and the lengths of the staple DNA were adjusted. By using the “Add Sequence” function, the base sequences were assigned to the designed structure. The designed DNA nanostructure was then rendered on an external website (Fig. 1d) [5]. In this hands-on session, the lecture focused only on two-dimensional structures, but three-dimensional structures can also be designed. Additionally, CanDo could simulate the thermal fluctuations of the designed structures [6].

Figure 1  Hands-on training of design of DNA origami. (a) The window of cadnano. (b) Making crossover. (c) Design of the trapezoidal DNA nanostructure. (d) The image of the trapezoidal DNA nanostructures.

At the end of the hands-on training, there was dedicated time for networking among the lecturers and participants. During networking, I had the opportunity to communicate with researchers from various backgrounds, allowing for more in-depth discussions about research, not only on DNA nanotechnology and biophysics. Most importantly, I was able to build a network with researchers from diverse research backgrounds.

At IUPAB2024, the session regarding the Hands-on training of DNA nanomachines was also held. While the Hands-on training focused mainly on the history and basic knowledge of DNA nanotechnology, the IUPAB sessions provided various latest research topics related to DNA nanotechnology. Learning the fundamentals of DNA nanotechnology and having experience designing DNA nanostructures during the hands-on training helped me better understand the presentation of this session. Prof. Endo showed that the various application types of DNA nanostructures include DNA nanostructures as templates for molecular assembly and the development of three-dimensional DNA nanostructure molecular devices capable of mechanical motion [7]. Then, Prof. Kuzuya introduced molecular devices that can open and close, like pliers, and recognize and capture specific molecules [8]. In addition, the integration of DNA nanotechnology into the synthetic molecule has been proposed. Prof. Suzuki introduced the lattice-shaped DNA nanostructure that can mechanically change their structure based on input DNA, and various types of changing structures were successfully achieved [9]. These presentations demonstrated that DNA can construct structures from the nanoscale to the microscale and add functionalities. Furthermore, the development of simulation software for DNA nanotechnology, the chromatin-like DNA nanostructure, and the nanopore sensing with the DNA nanostructure was reported. This session provided various insights and suggestions for the possibility of using DNA nanotechnologies.

In conclusion, by participating in hands-on training and the session at IUPAB2024, I was able to learn about the history and the latest research topics in DNA nanomachines (Fig. 2). Experiencing the DNA origami design software firsthand deepened my understanding of DNA nanomachines, and I am interested in incorporating it into my research. Even though the research field is different, it was a meaningful and enjoyable time. Constructing new networks with various researchers with the same research interests would especially be helpful for collaborative research in the future. Again, I gratefully thank the lecturers, planners, and everyone involved.

Figure 2  Certificate

References

• [1]   Seeman,  N. C. Nucleic acid junctions and lattices. J. Theor. Biol. 99, 237–247 (1982). https://doi.org/10.1016/0022-5193(82)90002-9
• [2]   Winfree,  E.,  Liu,  F.,  Wenzler,  L. A.,  Seeman,  N. C. Design and self-assembly of two-dimensional DNA crystals. Nature, 394, 539–544 (1998). https://doi.org/10.1038/28998
• [3]  Paul W. K. Rothemund. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006). https://doi.org/10.1038/nature04586
• [4]  cadnano. https://cadnano.org/
• [5]  DNA helix drawer. https://ibuki-kawamata.org/DNAHelixDrawer/
• [6]  CanDo. https://cando-dna-origami.org/
• [7]   Masubuchi,  T.,  Endo,  M.,  Iizuka,  R.,  Iguchi,  A.,  Yoon,  D. H.,  Sekiguchi,  T., et al. Construction of integrated gene logic-chip. Nat. Nanotechnol. 13, 933–940 (2018). https://doi.org/10.1038/s41565-018-0202-3
• [8]   Kuzuya,  A.,  Sakai,  Y.,  Yamazaki,  T.,  Xu,  Y.,  Komiyama,  M. Nanomechanical DNA origami ‘single-molecule beacons’ directly imaged by atomic force microscopy. Nat. Commun. 2, 449 (2011). https://doi.org/10.1038/ncomms1452
• [9]   Watanabe,  K.,  Kawamata,  I.,  Murata,  S.,  Suzuki,  Y., Multi-reconfigurable DNA origami nanolattice driven by the combination of orthogonal signals JACS Au, 3, 5, 1435–1442 (2023). https://doi.org/10.1021/jacsau.3c00091