Introduction of Intelligent Molecules lab.

The world's first dynamic DNA origami device born in Japan

Dynamic DNA origami devices were developed for the first time by Prof. Akinori Kuzuya of Kansai University, Osaka, Japan. DNA origami is a technique of utilizing DNA, a recording medium for the genetic information of all living organisms, as a building block to control structures and functions in a variety of ways through the self-assembly process. In addition to its strength in synthesizing raw materials, Kuzuya's lab is equipped with a range of devices that allow high-resolution visualization of prepared DNA origami at the single-molecule level and provides an experiment environment to validate its biological effects, which all contributed to the successful achievement. This article will give you a brief overview of the specific lab environments, as well as the potential of medical materials and devices created at his lab.

In-house synthesis of DNA raw materials

Generally, DNA raw materials for research are purchased from commercial reagent suppliers. However, the range of commercially available reagents is limited, which consequently limits the types of DNA origami that can be produced. The lab has a full range of equipment that enables in-house synthesis of DNA raw materials.

Kuzuya explains the advantages:
"Our lab is capable of synthesizing DNA raw materials that are needed for research, which means that we can obtain raw materials with special structures that do not exist yet today. In DNA origami, many short single-stranded DNAs (staples) fold a long single-stranded ring DNA (scaffold) into a molecular device with various shapes and functions. Our lab can synthesize DNA raw materials at will, and it is like a trading company for DNA products. This is one of the notable competitive advantages that distinguishes us from other top research groups in the world."

Feasibility as medical materials

Taking advantage of the lab's capability to synthesize from DNA raw materials, Prof. Kuzuya introduced the world's first pliers-like dynamic DNA origami device to the field of DNA research, which only had techniques to produce static, fixed-shape nanostructures until then. Upon finding a target molecule, this DNA origami executes the function to capture the target, similar to using a pair of pliers to grab an object. The research team has been working jointly with a medical IT venture to commercialize a chemical test kit (patent pending), with the aim of developing products such as cancer diagnostic kits to detect chemicals that are released by cancer patients.

Kuzuya's other research interests include DNA quadruplex gel (Chem. Asian J., 2017, 12, 2388). DNA quadruplexes trap ions such as potassium and sodium, which promote formation of quadruplex aggregates. The research team created a new gel—DNA quadruplex additionally reacted with highly biocompatible ethylene glycol chains—which solidifies instantly upon detecting salinity in the body such as tear drops and saliva.

a) Structure of PEG–DNA conjugates used in this study. Photograph: purified L4.6k-dG4 (1 g) prepared by modified HELP (high-efficiency liquid phase) synthesis.

b) Schematic representation of the system. Wavy blue lines represent PEG segments. Upon the addition of K+ or Na+ to a conjugate solution, DNA segments immediately form G-quadruplexes to crosslink PEG segments into a 3D network.

c) Photographs of 10 wt % DNA-PEG-DNA conjugate in 0.2 m Tris/HCl before (left) and after (right) the addition of metal ion solutions (100 mm K+ or 100 mm Na+, final concentrations).

This technology is anticipated to be utilized for drug delivery vehicle and other applications. DNA quadruplex gel is one of the research topics for the medical-engineering collaboration project "Kansai University Smart Materials and Advanced Therapeutics (KU-SMART)," which was adopted by the Ministry of Education, Culture, Sports, Science and Technology and has been implemented since FY2016. It is an outstanding research project of Kansai University that is gathering attention in Japan.

Thanks to KU-SMART, Kuzuya's lab is furnished with equipment to carry out in vivo functional evaluation. The new equipment allows the validation of the effects of medical materials under development, which constitutes another significant advantage in further promoting the research.

A range of exceptional equipment in the world that accelerates research

Prof. Kuzuya's research requires a range of high-quality equipment to directly observe and verify whether the created molecular devices or materials function exactly as planned.

Kuzuya points out:
"In DNA origami research, it is essential to observe the synthesized DNA structure to ensure that it functions as designed. Our lab has a range of equipment optimal for observing structures at the single-molecule level."

One such equipment is the atomic force microscope (AFM), which can be used to observe the morphology of molecules. The lab has three sets of AFM, two of which are high-speed AFMs that can take images at a rate of 10 frames per second. This makes it possible to observe the dynamic movement of molecules by connecting consecutive still images, which is not possible with standard-speed AFM. Kuzuya explains the competitive advantage of utilizing high-speed AFM videos for observation:
"High-speed AFM provides real-time imaging of the movement of molecules, allowing us to observe the way they move as designed and to obtain the evidence. This is one of the outstanding strengths of our lab."

Other notable pieces of equipment at Kuzuya's lab include a confocal fluorescence microscope, which is capable of 3D imaging. This system has an advantage of a measurement speed higher than that of other confocal microscopes, and it can also film 3D movies. It is therefore essential for his research on dynamic DNA origami. The confocal fluorescence microscopes are fitted with a camera for super-resolution image processing, enabling high-resolution imaging beyond the diffraction limit of light.

The lab also has total internal reflection fluorescence microscopes, which are suitable for observing single molecules, allowing researchers to observe DNA origami at the single-molecule level.

Kuzuya describes the exceptional research environment:
"It is not uncommon for labs to have a set of AFM or a set of fluorescence microscope. However, I do not think there are many labs in the world that have multiple sets of both types of microscopes, allowing researchers to use specific microscopes for specific purposes at one location like our lab. Furthermore, this environment can be used not only for DNA origami research, but also for the broad field of nanotechnology, such as metal nanoparticles and quantum dots."

Lab opened to researchers from all over the world

Prof. Kuzuya receives many offers to conduct joint research from researchers and research groups from all over the world owing to his exceptionally well-equipped research environments and accomplishments in the field of DNA origami research from the early stages. Recently, Kuzuya conducted studies jointly with Yoko Yamakoshi of the Swiss Federal Institute of Technology in Zürich (ETHZ) with results published in papers (ACS Nano 2021, 15 (12), 19256–19265). Currently, Dr. Qing Liu from the Chinese Academy of Sciences has joined Kuzuya's lab and has been conducting joint research as a postdoctoral fellow of Kansai University. Qing Liu outlines his research:

"My research topic is two-fold: one is to observe the DNA double-helix structure at a high-resolution using AFM to obtain the DNA sequencing information, and the other is to change the DNA origami structure using the AFM Probe tip to measure directly the mechanical properties of DNA origami molecules, including the hardness and force required to change the structure."

Qing Liu's research is supported by the research grant program of the New Energy and Industrial Technology Development Organization (NEDO), and he has been accepted for Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowships for Research in Japan.

The research environment of Kuzuya's lab, which has a range of equipment to carry out DNA raw material synthesis, structural observation, and in vivo functional evaluation, is attractive for researchers involved in a broad range of scientific research, including bioorganic chemistry and synthetic organic chemistry, in addition to DNA origami. Furthermore, Kansai University is one of the more progressive universities in Japan that responded early to the COVID-19 outbreak. Kuzuya also promptly started COVID-19 research under the framework of the project of "Development of intranasal COVID-19 vaccine using macromolecule micelles."

Lastly, Kuzuya says:
"If you have a research project that you have not implemented, but believe that you can do it in a research environment with a range of the newest equipment, join us and use our lab equipment. We are located in Osaka, which is one hour away from Kyoto or Nara by train. This makes the environment perfect not only for research, but also for experiencing traditional Japanese culture. We welcome all researchers from around the world to contact us."

Reference

Kuzuya, A., Sakai, Y., Yamazaki, T., Xu, Y. & Komiyama M. Nat Commun 2, 449 (2011).

Tanaka, S., Wakabayashi, K., Fukushima, K., Yukami, S., Maezawa, R., Takeda, Y., Tatsumi, K., Ohya, Y., & Kuzuya. A. Chem. Asian J, 12, 2388 –2392 (2017).

Ray, A., Passiu, C., Nasuda, M., Ramakrishna, S. N., Rossi, A., Kuzuya, A., Spencer, N. D., and Yamakoshi, Y. ACS Nano 2021 15 (12), 19256–19265, (2021).

Intelligent Molecules Lab., Kansai University

KU-SMART Project, Kansai University

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Email: kansai-u1886ml.kandai.jp