Akio Nishikawa

Robust and photocontrollable DNA capsules using azobenzenes.

Various three-dimensional structures have been created on a nanometer scale using the self-assembly of DNA molecules. However, ordinary DNA structures breakdown readily because of their flexibility. In addition, it is difficult to control them by inputs from environments. Here, we construct robust and photocontrollable DNA capsules using azobenzenes. This provides a method to construct DNA structures that can survive higher temperatures and can be controlled with ultraviolet irradiation.

In vitro maturation of collagen fibrils modulates spreading, DNA synthesis, and collagenolysis of epidermal cells and fibroblasts☆

Collagen fibrils were maturated in vitro by incubating them in a serum-containing culture medium at 37 degrees C for varied lengths of time. Epidermal cells and fibroblasts were cultured on these maturated collagen gels to see the effects of maturation on cellular morphology and physiology. The spreading and DNA synthesis of both types of cells on the maturated collagen gels were significantly enhanced compared to those on fresh gels. The maturation did not affect the cellular adhesiveness to the substrate. The secretion of collagenase by epidermal cells was suppressed on the maturated collagen gels, the extent of the suppression being related to the length of maturation of the gels. These maturation-related effects of collagen were also observed when collagen was incubated in the medium without serum, indicating that the effects are not due to deposition of serum proteins to collagen gels during maturation. Physical and chemical characterizations of the maturated collagen were performed: the mechanical strength of collagen gels increased in maturated collagen gels, the amounts of insoluble collagen increased with the maturation. These changes in the chemical and physical nature of the maturated collagen gel strongly suggested that there was an increase in intermolecular crosslinks during the process of maturation. These maturation-induced changes in collagen were marked when collagen gels were incubated in the presence of glucose, indicating that a glucose-protein reaction such as the Maillard reaction is involved in this phenomenon.

DNA computation simulator based on abstract bases

Abstract We developed a simulator to aid those who design algorithms and protocols for DNA computing. In this simulator, abstract sequences instead of real DNA sequences are used to represent molecules in order to increase efficiency of simulations. Two approaches for simulation are available: threshold and stochastic. The simulator consists of two main parts, one for finding reactions among existing molecules and generating new ones, and the other for numerically solving differential equations to calculate the concentration of each molecule. The two parts rely on each other. In particular for the threshold approach, the former avoids a combinatorial explosion by setting a threshold on concentrations of molecules that can take part in reactions. In addition, the stochastic approach is also available for simulations which are hard by the threshold approach. Some simulation results by the approaches are also presented: computation of Boolean circuits, whiplash PCR, formation of DNA tiles and polymerase chain reaction (PCR). We also integrate simulating DNA computation and fitting parameters by the genetic algorithm (GA), where simulation results are used as evaluation functions for the genetic algorithm. The integration is applied to find good protocols for PCR amplification. A trial to refine the reaction model for hybridization is also described before the final discussion on the simulator.

DNA-based crosstalk nanorobot mimicking amoeba type of slime funguses

Toward the realization of the intelligent control of internal and external environments, we have presented a DNA-based nanorobot that can communicate with bacteria. This nanomachine has the potential ability to recognize signals of bacterial cell-cell communication called quorum sensing and produce antibiotic aptamers. We have succeeded in demonstrating that the synthetic DNA robot mimicking slime funguses can serve an effective ON-OFF control system for the aptamer transcription in response to quorum sensing signals. Our results indicate that the DNA nanotechnology can provide promising nanometer-sized machines for the effective control of the bacterial population balance by the appropriate antibiotic aptamer transcription.

Design of molecular-based network robots-Toward the environmental control

Toward the intelligent control of internal and external environments, we present an idea of the sequence design for the network formation of DNA-based nanorobots. We already developed a DNA nanomachine that has the potential ability of producing a functional molecule called “aptamer” when bacterial two-component signal transduction systems are detected. Here we have succeeded in demonstrating the network formation based on the sticky-end programming of DNA modules. By combining the aptamer transcription function with the network formation ability, we can implement various missions in DNA nanomacines. The network design is essential to keep the appropriate density, configurations and functions to carry out target missions. We expect that the network of DNA nanorobots make a contribution to realize the effective regulation of microorganisms to promote environmental preservation and health maintenance.

Regulation of desmin expression in adult-type myogenesis and muscle maturation during Xenopus laevis metamorphosis.

Isoforms of myosin heavy chain and tropomyosin convert during metamorphosis of Xenopus laevis with larval-to-adult remodeling of dorsal muscle (Nishikawa and Hayashi, 1994, Dev. Biol. 165: 86–94). In the present study, other markers for muscle remodeling during metamorphosis were determined by SDS-PAGE analysis. The amounts of twelve muscle proteins changed remarkably during metamorphosis. Among these, a 54-kDa molecule was found to be desmin, and the relative content/total proteins decreased dramatically through metamorphosis. In hindlimb muscle, desmin content increased fourfold during prometamorphosis, when myoblast proliferation and fusion occurred. With further myotube maturation, this content decreased by 1/2 while that of muscle actin continued to increase. Thus, desmin up- and down-regulation in hindlimbs mark early and late phases of myogenesis, respectively. In tail muscle, the desmin content decreased continuously to 1/8 before and during metamorphosis, due to tail muscle growth and maturation. In dorsal muscle, three desmin changes occurred: a pre-metamorphic decrease, a transient increase at prometamorphosis, and a rapid decrease at the climax stage. Immunohistochemical analysis showed desmin+ cells to be present between young (adult-type) myotubes and replicating (PCNA+) cells in dorsal muscles, correlating the transient desmin upregulation in dorsal muscle with the initiation of adult-type myogenesis. After the upregulation, dorsal muscle desmin decreased to 1/8. This rapid down-regulation was replicated by administration of triiodothyronine (T3) to tadpoles, suggesting a significant role for T3 in dorsal muscle remodeling during metamorphosis. Collectively, these results show that analysis of desmin expression and PCNA-immunohistochemistry are good tools for determining the sites and timing of larval-to-adult muscle remodeling during Xenopus laevis metamorphosis.

Implementation of tile sequencing for DNA logical computation toward next-generation information security

We present a new readout method for DNA-based logical computation. It is expected that DNA computing can offer effective solutions to difficult-to-solve problems for the conventional computing. On the other hand troublesome procedures to read out the calculation results prevent the realization of practical applications of DNA computing. Here we propose a new tile sequencing method to read out the DNA tile computing results using a visualization tile. We verify its effectiveness by applying to DNA-based one-time pad (OTP) cryptosystem and clarify the problems to be resolved to provide the practical applications. It is known that OTP serves a theoretically-unbreakable cryptosystem if the random key is truly random, never reused, and kept secret. DNA hybridization process can be a physical random source required for true-random-key generation. Because a mathematical algorithm can generate pseudo-random numbers only, DNA computing will realize a next-generation cryptosystem that integrates a self-supported true random number generator. Our results indicate that the tile sequencing will be able to provide a new label and amplification free convenient high-throughput readout system for DNA tile computing, when it is combined with the multi-layer algorithm. Our new readout method will promote the progress of DNA logical computation and expand the possibility of its practical applications.

Formation of a New Limb Bud at the Boundary Between a Transplanted Limb Bud and the Tail Surface of Xenopus Tadpoles

Through transplantation experiments with Xenopus laevis tadpoles, we found a new morphogenetic phenomenon consisting of limb bud formation at the boundary between transplanted whole limb buds and the tail surface. This phenomenon occurs without limb—limb stump interaction and has a number of unique features: (1) Only one extra limb bud was formed per transplant and the new limb and the transplanted limb were bilaterally symmetrical, forming a pair of limb girdles. (2) Extra new limb bud formation occurred not only at the tail but also at other non-limb regions, including abdominal and head surfaces. (3) Successful limb formation required the presence of basal 1/4 region (presumptive limb girdle) of a limb bud explant. (4) New limb formation was host-stage-dependent: before metamorphosis, limb bud formation ratio was high (> 90%), but as the host tadpole entered metamorphosis, this potential declined and morphological abnormalities of new limbs increased. (5) Cell lineage analysis showed that epidermis of the new limb bud always contained many (about 60%) host-derived cells, while new limb cartilage cells were completely graft-derived. These results suggest that heterotopic new limb formation occurs through interaction between graft mesenchyme and host epidermis. Thus, the present study has clarified the two important aspects of limb ontogeny: the importance of presumptive limb girdle for the limb bud initiation and the relationship between limb bud formation potential and metamorphic tissue remodeling. The present experimental system may help to improve our understanding of epithelial-mesenchymal interaction during limb bud initiation and subsequent limb cell differentiation during metamorphosis.

Multi-fueled approach to DNA nano-robotics

An approach to multi-fueled DNA nano-robotics is described. We propose three types of driving force (i.e., fuel for DNA nano-robots): thermal fuel, pH fuel, and light fuel. The thermal fuel controls the hybridization of DNA molecules around the melting temperature. The pH fuel controls the hybridization of the so-called i-motif by changing the pH condition. The light fuel controls the hybridization of DNA oligomers that are intercalated with azobenzene by irradiation with UV or visible light. These three fuels are not mutually exclusive. However, experimental conditions for the fueling of DNA nano-robots show efficacy. Concrete ideas for using these three fuel types are proposed and discussed. In addition, the results of calibration experiments and preliminary results for refining pH fuel sequence are also shown.

Towards Multi‐fueled DNA walker on DNA trails

Basic idea and preliminary experimental results for photo‐controllable DNA tiles are described. Although self‐assembly of DNA cross tiles is an established technique, it only results in a static structure controlled by sequence design of sticky ends. For realizing dynamic control of DNA nanostructures, multi‐fueled approach to self‐assembly of DNA cross tiles is introduced. It is based on thermal‐fuel, pH‐fuel and photo‐fuel. We already verified their basic behaviors in test tubes. In addition, we started to examine them for DNA walker on DNA trails made of DNA cross tiles. Especially, azobenzene intercalation groups for the sticky ends in DNA cross tiles are useful for photo‐fuel, as they can control the assembly process with irradiation of UV/visible light. Preliminary results concerning the DNA cross tiles with azobenzene are also described briefly.