DNA Nanotechnology Research in Japan

Abstract

In this essay, the evolution of DNA nanotechnology research in Japan to date will be reviewed. The expansion of the research community in Japan and the trends in regard to the selection of project themes will be elucidated, along with the identification of the researchers who participated in these projects. Some aspects of the research history of the author, who entered from the field of robotics, are introduced, as this information may be of interest to young students and researchers.

1 Introduction

In 1982, when Professor Seeman began his research on DNA nanostructures, there were no studies of this kind in Japan. In 1994, Adleman’s work on DNA computers was published, and two years later, Masami Hagiya of the University of Tokyo initiated a research project on molecular computing [1]. Using this as a starting point, the history of DNA nanotechnology in Japan has evolved over approximately 25 years. Hagiya would then lead research in Japan for the next 20 years. Figure 1 shows the genealogy of related projects in Japan.

Fig. 1

DNA nanotechnology-related projects in Japan

Early projects explored the possibility of massively parallel computation with molecules, and in 2001, a project on molecular programming [2] began, which included researchers from disciplines such as mechanical, materials, and medical engineering. These scientists introduced the idea of extending the function of the system beyond “computation,” to sensing of the environment and acting accordingly. After several years of preparation, the project of “molecular robotics” was launched in 2012 [3].

There are several reasons why the concept of “molecular robots” originated in Japan. One is that Japan is a country where robotics research is very active, and there are a large number of researchers, in both the theoretical and applied fields. There is also a latent familiarity with the word “robot” through various depictions in cartoons and animations. On the other hand, research in chemistry, especially organic chemistry and polymer science, is also active in Japan, as is evidenced by the fact that the country has produced a number of Nobel laureates in chemistry. Many researchers in these fields are working on specific applications, while others are attempting to create new molecules out of scientific curiosity, with the expectation that the application of their molecules will be beneficial in the future. The author believes that these types of researchers may have been attracted by the concept of “building robots with molecules.”

During the five years of the project, several prototypes have been developed. The amoeba-type molecular robot contained various molecular devices that were encapsulated in artificial cells. In the gellular automaton, programming of a molecular computing system on a gel medium was investigated. In parallel with the progress of the project, a community of researchers in molecular robotics was organized as an arm of the Society of Instrument and Control Engineers (SICE), and research workshops and national conferences were held regularly. In 2020, a project on “molecular cybernetics” was launched, with the author as its representative [4]. The goal of this project was to create a system with a certain information-processing capabilities by combining artificial cells developed through molecular robotics.

2 How the Author Got Involved in DNA Nanotechnology

The author specializes in robotics, specifically autonomous distributed robotic systems (DARS) [5]. Research on these systems focuses on flexibility and adaptability, which is not possible with conventional robots, through the coordination of a large number of autonomous robotic agents. The author is particularly interested in “homogeneous” autonomous distributed robot systems, that is, systems consisting of a large number of robot modules of exactly the same type. Each module is a small robot with a microcomputer, sensors, and actuators. (This type of robot is therefore called a modular robot.) Similar to building a house with Lego blocks, the modules not only connect with each other to form different shapes, but also move and recombine themselves to change the shape of the whole structure without external aid. My group proposed the concept of such modular robots and developed several prototypes to demonstrate this concept [6]. For example, we have created a “self-repairing” robot that can automatically replace a failed module with a spare one, and a modular robot that can autonomously transform itself into different forms, such as that of a dog or a snake, and move according to these forms.

Several other groups have also developed homogeneous distributed robots, but the number of modules was limited to between 10 and 100. Therefore, these prototypes had reduced functionality. There are various reasons why the number of modules cannot be increased (i.e., difficulty in creating a scalable system). For instance, each module must contain such things as a microcomputer, sensors, motors, batteries, and communication devices in a compact space, which is difficult to design in itself; it is challenging to guarantee mechanical, electrical, and information reliability, and, when possible, it comes with a large cost. Modular robots are expected to be used for exploration and rescue in unknown environments because of their ability to adapt to the conditions, but full-scale applications would require thousands or tens of thousands of modules, which is hindered with current technology. The Kilobot developed by Nagpal et al. is a system with 1000 modules and is probably the largest modular robot built to date [7]; however, each module is only able to move on a plane, and they cannot be connected to each other.

In 2000, when I moved from the National Mechanical Engineering Laboratory (MEL) to the Tokyo Institute of Technology, I was unsure if I would continue my research on modular robots. I had been simulating the formation of networks of various shapes using a model in which mass points were connected by virtual springs [8]. I learned that it is possible to make a jigsaw puzzle of molecules using DNA, that is, they could self-assemble [9]. I had always been interested in the term “self-assembly” and even called my modular robot a “self-assembling machine” [10]. Therefore, I began studying DNA tiles with my students and developed a method to improve the reliability of algorithmic self-assembly, which had been proposed by Winfree and others at that time [11]. Subsequently, I learned directly from Professor Winfree and gradually entered this field.

Many mechanical engineers have changed their research subjects significantly in the course of their research life. My personal impression is that approximately 50% alter their research interests at some point. Professor Klavins at the University of Washington is another scientist who moved from the field of modular robots to that of bio/nanotechnology. He has been working on modular robots that operate stochastically [12] (his system of randomly moving pizza piece-like modules on an air-hockey table to make perfectly round pizzas is fascinating to watch). He is now a synthetic biologist.

Young people who are about to start their research should be prepared for the possibility that they may completely change their research field in the future. This does not mean starting the research from scratch, as the methodologies acquired up to that point are often applicable for different research areas. Changing their focus expands their futures by broadening the range of methodologies available to them.

3 The Evolution of Projects in Japan

Let us return to the research projects in Japan. First, I would like to briefly explain the scientific research system in Japan. Most basic research in the sciences, engineering, and humanities is funded by public grants from the government. The main subsidy is a Grant-in-Aid for Scientific Research (KAKENHI) provided by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) [13]. In addition, the Japan Science and Technology Agency (JST), affiliated with MEXT, and the New Energy and Industrial Technology Development Organization (NEDO), affiliated with the Ministry of Economy, Trade and Industry (METI), also provide large research funds, but these funds are only for application-oriented projects.

In the field of DNA nanotechnology, KAKENHI is the main source of funding. There are various levels of KAKENHI, ranging from a few million yen per year for a small number of researchers to several hundred million yen per year for a few dozen researchers. All of these are competitive research funds, and each obtained with different levels of difficulty. As expected, projects with larger budgets have more difficulty obtaining these grants, and often the largest schemes are awarded after years of repeated applications. In particular, the KAKENHI scheme, which is awarded to groups of several dozen researchers (the scheme name has changed from “Priority Area Research” to “Innovative Area Research” to “Transformative Area Research,” but the content is similar), aims to establish a new academic community in the field. By following this transition, we can observe the trend of research in Japan.

3.1 The 1980s and 1990s

In the 1980s, DNA research in molecular biology was active in Japan, but no projects in relation to DNA nanotechnology had yet been initiated. DNA nanotechnology can be divided into two categories: structural DNA nanotechnology founded by Seeman, and DNA computing founded by Adleman, and considering these, DNA computing research actually began earlier in Japan. That is, two years after the first paper on DNA computing by Adleman [14], a research project on molecular computers (1996–2002) [1] was conducted under the leadership of Masami Hagiya at the University of Tokyo. Professor Adleman was well known in the field of computer science, and DNA computing was expected to be a massively parallel computing architecture that could outperform supercomputers. At that time, Takashi Yokomori (Waseda University) and others in the Japanese computer science community had obtained information about DNA computing through researchers in formal language theory. In other words, expectations of molecular computation were raised at a relatively early stage in Japan. Senior researchers Yuichiro Anzai, in computer science (Keio University) and Shigeyuki Yokoyama, in structural molecular biology (University of Tokyo), who were in a position to select new projects under the “Research for the Future Program”, chose Professor Hagiya, who had been working on a follow-up to Adleman’s experiment, as the leader of the project. The influence of both the bottom-up interest of young researchers and the top-down direction of senior researchers at the beginning of this field in Japan is evident.

The molecular computer project [1] started by Hagiya et al. was joined by Takashi Yokomori, Akira Suyama (University of Tokyo) in biophysics, and later by Masayuki Yamamura (Tokyo Institute of Technology) in computer science. The SAT engine using DNA hairpin formation and Whiplash PCR are among the results of the project. The SAT engine solves the satisfiability problem known for hard combinatorial problems by using the hairpin formation of DNA strands [15, 16]. Whiplash PCR combines the opening/closing of DNA hairpins with polymerase elongation to achieve state representation and state transitions in a single hairpin molecule; the term “whiplash” came into use subsequently [17, 18]. In the same year, 1996, the project of “Molecular Memory” was conducted under Hagiya [19]. In this project, Azuma Ouchi (Hokkaido University), Jun Tanida (Osaka University), and others in computer science participated, and various types of molecular memories were developed including “nested primer molecular memory” [20], which amplified only DNA with a specific sequence by four-level PCR, and a molecular memory that read information spatially by transduction between two secondary structures using the infrared laser excitation of hairpin DNA fixed on the substrate plane. Conformational addressing of multiple hairpin DNAs [21] was also developed in this project.

3.2 The 2000s

In 2002–2007, the Grant-in-Aid for Scientific Research on Priority Areas “Molecular Programming” was conducted, again with Hagiya as the representative [2]. This was one of the largest KAKENHI research projects, involving approximately 50 researchers and lasting five years. In contrast to the previous research on “molecular computation,” which aimed to synthesize a desired function or structure by utilizing the computational potential of biomolecules, “molecular programming” considered the process of designing biomolecules and their chemical reactions as “programming” and aimed to develop a systematic programming methodology for molecular computation. For this project, in addition to the members of the previous projects, Daisuke Kiga (Waseda University) in synthetic biology, Kenzo Fujimoto (Japan Advanced Institute of Science and Technology) in nucleic acid chemistry, and the present author (Tohoku University) in robotics were involved. The first half of the project focused on the refinement of DNA sequence design technology, and the second half on its application in nanotechnology and synthetic biology.

For this project, Suyama et al. developed a molecular computational reaction system (R-TRACS) using DNA and RNA enzymes [22], and Kiga et al. created a bacterial computer using E. coli [23]. In addition, the author worked on the DNA tile error rate evaluation [24] with Winfree. At the same time, the author attempted to grow DNA tiles in microfluidic devices with Teruo Fujii (now President of the University of Tokyo). In Fujii’s laboratory, Yannick Rondelez began studying the reaction system using DNA enzymes in 2009, and this developed into the PEN DNA toolbox [25].

Subsequently, frequent meetings have been held, mainly by those involved in the molecular programming project, and a community of researchers using DNA nanotechnology as a tool has been formed. In June 2002, the DNA8 workshop was held in Sapporo (Hokkaido University), hosted by the molecular programming project (Professor Seeman presented a keynote lecture).

3.3 The 2010s

After the end of the molecular programming project, proposals were made every year to obtain large project research funds, but none were accepted. In 2010, the author’s proposal, “Development of molecular robotics by DNA nanoengineering,” was selected as a relatively large project under the “Grant-in-Aid for Scientific Research (S)” scheme. Akinori Kuzuya (Kansai University), who studied DNA origami in Seeman's laboratory, and Shin-ichiro Nomura (Tohoku University) who was involved in artificial cell engineering participated in this project. This project introduced the term “molecular robotics,” which uses a DNA nanotechnology to design component molecules and assemble them to create a functional molecular system that can respond autonomously to changes in the environment. The “Molecular Robotics Research Group” was established in the Society of Instrument and Control Engineers (SICE) during this time. The author served as the representative of this group. This research group continues, and the majority of the researchers involved in DNA nanotechnology and DNA computing in Japan have participated.

In 2011, Grant-in-Aid for Innovative Field “Synthetic Biology” was implemented (2011–2016) with Masahiro Okamoto (Kyushu University) as the representative. Yamamura, Kiga, and Suyama, who participated in the molecular programming, moved to this group.

In 2012, the project “Molecular Robotics—Creation of molecular robots with sensation and Intelligence” was adopted as Innovative Area, led by Hagiya [3]. The core members of this project were a combination of computer science researchers (Hagiya, Satoshi Kobayashi (University of Electro-Communications), Akihiko Konagaya (Tokyo Institute of Technology), and experimental and implementation researchers (the author and Hirohide Saito (Kyoto University), who was in the field of RNA nanotechnology).

Generally, a “robot” can be defined as an “autonomous system” that acquires information from the external environment using sensors, processes this information, and acts on the environment accordingly. A body (structure) is also required to distinguish the system from the environment and to integrate these components. The goal of the project was to develop technologies for the design, fabrication, integration, and control of molecular robots.

An evolutionary scenario for molecular robots was proposed at the inception of the molecular robotics project (Fig. 2) [26]. This scenario predicts the gradual evolution of molecular robots over four stages: Generation 0: A single molecule acts as a robot. Its behavior is random due to thermal fluctuations. This would be similar to the molecular spider of Stojanovic et al. [27]; Generation 1: Artificial cell membranes encapsulating various molecular devices. When several kinds of molecules are enclosed in a container, the concept of “concentration” arises, and its behavior can be predicted to some extent by chemical reaction kinetics. This is called an amoeba-type molecular robot; Generation 2: Various molecular devices are dispersed in a gel medium, whereby each molecule has a spatial distribution, resulting in a system with information on both “concentration” and “position.” This is called a slime-type molecular robot; Generation 3: The molecular robot of Generation 1 is multicellularized. Multicellular robots can be realized with more complex and diverse functions; and Generation 4: Hybridization, in which the molecular robot is expected to be combined with conventional nanotechnology such as photolithography.

Fig. 2

Evolutionary scenario of molecular robotics

In the molecular robotics project, the goal was to achieve the first and second generations of this scenario. For Generation 1, the development of an amoeba-like molecular robot, we succeeded in constructing a system combining a light sensor, a DNA amplification circuit, a DNA molecular clutch, and a microtubule-kinesin molecular motor in a single artificial cell (liposome). We demonstrated that the liposome deforms like an amoeba and turns on and off using light stimuli [28]. For Generation 2, the slime-type molecular robot, a combination of BZ gel actuator (a self-oscillating gel actuator using the Belousov-Zhabotinsky reaction) and DNA computing was initially planned, but the operating condition of the BZ gel actuator required strong acidity, which is incompatible with the operating conditions for DNA, and the project was abandoned. Instead, the molecular implementation of a cellular automaton system in gel space, called a gellular automaton, was explored [29, 30].

In addition to amoeba-type molecular robots, various technologies have been developed. For example, RNA nanostructure devices have been developed as therapeutics. They function under the strong noise of various biomolecules and successfully control the fate of cancer cells [31]. Another example is a technique to speed up various DNA computational reactions by combining synthetic DNA and artificial nucleic acids [32]. Furthermore, microtubule assemblies controlled by light-responsive DNA [33] and gel actuators driven by DNA-controlled gel-sol phase transition [34] have been developed as actuators for molecular robots.

The results of the “molecular robotics” project were summarized in a textbook, “Introduction to Molecular Robotics” [35], which was published in Japanese. The English version of the textbook is currently being edited. In 2014, DNA20 was held in Kyoto (Kyoto University), hosted by the molecular robotics project. The conference included a keynote lecture by Professor Seeman on “Molecular machines made from DNA.”

In the year following the completion of the molecular robotics project, two new KAKENHI projects, as Innovative Areas, were launched, namely “Molecular Engine” (2018–2023), led by Kazu Kinbara and “Creation of Soft Robotics” (2018–2023), led by Koichi Suzumori, both of whom were from the Tokyo Institute of Technology. These were the research areas of active matter and soft robotics, respectively. Some of the members of Molecular Robotics have participated in these projects, and collaboration between these areas is progressing.

3.4 Current Research

Four years after the molecular robotics project, a project entitled “Molecular Cybernetics—Construction of a minimal artificial brain by the power of chemistry” was adopted as a KAKENHI Transformative Area (A) [4]. Transformative Areas (A) are similar to Innovative Areas. The Molecular Cybernetics is led by the author with 18 core researchers including Taro Toyota (University of Tokyo) and Shin-ichiro Nomura, Akinori Kuzuya, and Takashi Nakakuki (Kyushu Institute of Technology) in control engineering.

The amoeba-type molecular robot can have a variety of molecular devices implemented in a single artificial cell (liposome). However, it is difficult to derive appropriate solution conditions under which all the different molecular species, such as light-responsive artificial nucleic acids, DNA devices, and molecular motors, can function. To solve this problem, we use multiple liposomes that contain solutions suitable for each of these molecular devices and bind them together to build a system. The liposomes are connected by a “transducer” for communication, which transmits molecular information without mixing the solutions. Our goal was to realize a simple learning function on a three-liposome system called a minimal artificial brain (Fig. 3).

Fig. 3

Minimal brain concept in molecular cybernetics project

In molecular cybernetics, there are several new approaches that have yet to be attempted. One of these is a variety of free services for members of the group. Kazunori Matsuura of Tottori University (peptide engineering) runs a peptide synthesis center that provides various peptide chains upon request from the members of the project. Keiji Murayama of Nagoya University (nucleic acid chemistry) runs the nucleic acid synthesis center, which provides nucleic acid sequences containing special artificial bases. In addition, Kuzuya of Kansai University will provide a range of single-molecule observation services using various microscopes and atomic force microscopes. Another new activity of the project is journalist-in-residence. Mikihito Tanaka of Waseda University (social analysis) hosts external non-specialists, such as newspaper journalists, science writers, and science fiction authors. They will help society to accept the advanced concept of molecular cybernetics by providing an objective view of the ongoing project and disseminating information as links between researchers and the general public. In 2023, DNA29 will be held in Sendai (Tohoku University), hosted by the molecular cybernetics project.

4 Summary

This essay describes the history of the development of the research community in Japan, focusing on the evolution of research projects conducted to date. In addition, an example of how researchers can become involved in an emerging field such as DNA nanotechnology is given through the author's personal research history. Currently, the COVID-19 pandemic continues to be unpredictable (January 2022). In Japan, the spread of the sixth wave is now feared, and almost all communication between researchers is restricted to online contact only. Once the pandemic ends, we will again be able to freely hold conferences and more readily collaborate to bring this research into the future.

Professor Seeman passed away during the editing of this manuscript. He first revealed the possibility of molecular robotics based on DNA nanotechnology, and many researchers in Japan have been directly or indirectly influenced by him. I would like to express my gratitude for his guidance and provide my sincere condolences on his loss.