Atsushi Kameda

A novel ATP regeneration system using polyphosphate-AMP phosphotransferase and polyphosphate kinase

Polyphosphate-AMP phosphotransferase (PAP) and polyphosphate kinase (PPK) were used for designing a novel ATP regeneration system, named the PAP-PPK ATP regeneration system. PAP is an enzyme that catalyzes the phospho-conversion of AMP to ADP, and PPK catalyzes ATP formation from ADP. Both enzymes use inorganic polyphosphate [poly(P)] as a phosphate donor. In the PAP-PPK ATP regeneration system, ATP was continuously synthesized from AMP by the coupling reaction of PAP and PPK using poly(P). Poly(P) is a cheap material compared to acetyl phosphate, phosphoenol pyruvate and creatine phosphate, which are phosphate donors used for conventional ATP regeneration systems. To achieve efficient synthesis of ATP from AMP, an excessive amount of poly(P) should be added to the reaction solution because both PAP and PPK consume poly(P) as a phosphate donor. Using this ATP generation reaction, we constructed the PAP-PPK ATP regeneration system with acetyl-CoA synthase and succeeded in synthesizing acetyl-CoA from CoA, acetate and AMP. Since too much poly(P) may chelate MG2+ and inhibit enzyme activity, the Mg2+ concentration was optimized to 24 mM in the presence of 30 mM poly(P) in the reaction. In this reaction, ATP was regenerated 39.8 times from AMP, and 99.5% of CoA was converted to acetyl-CoA. In addition, since the PAP-PPK ATP regeneration system can regenerate GTP from GMP, it could also be used as a GTP regeneration system.

Destruction of Amyloid Fibrils of a β2-Microglobulin Fragment by Laser Beam Irradiation

To understand the mechanism by which amyloid fibrils form, we have been making real-time observations of the growth of individual fibrils, using total internal fluorescence microscopy combined with an amyloid-specific fluorescence dye, thioflavin T (ThT). At neutral pH, irradiation at 442 nm with a laser beam to excite ThT inhibited the fibril growth of β2-microglobulin (β2-m), a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Examination with a 22-residue K3 fragment of β2-m showed that the inhibition of fibril growth and moreover the destruction of preformed fibrils were coupled with the excitation of ThT. Several pieces of evidence suggest that the excited ThT transfers energy to ground state molecular oxygen, producing active oxygen, which causes various types of chemical modifications. The results imply a novel strategy for preventing the deposition of amyloid fibrils and for destroying preformed amyloid deposits.

A separation method for DNA computing based on concentration control

A separation method for DNA computing based on concentration control is presented. The concentration control method was earlier developed and has enabled us to use DNA concentrations as input data and as filters to extract target DNA. We have also applied the method to the shortest path problems, and have shown the potential of concentration control to solve large-scale combinatorial optimization problems. However, it is still quite difficult to separate different DNA with the same length and to quantify individual DNA concentrations. To overcome these difficulties, we use DGGE and CDGE in this paper. We demonstrate that the proposed method enables us to separate different DNA with the same length efficiently, and we actually solve an instance of the shortest path problems.

Chain reaction systems based on loop dissociation of DNA

In the field of DNA computing, more and more efforts are made for constructing molecular machines made of DNA that work in vitro or in vivo. States of some of those machines are represented by their conformations, such as hairpin and bulge loops, and state transitions are realized by conformational changes, in which such loops are opened. The ultimate goal of this study is to implement not only independent molecular machines, but also networks of interacting machines, called chain reaction systems, where a conformational change of one machine triggers a conformational change of another machine in a cascaded manner. A chain reaction system would result in a much larger computational power than a single machine in the number of states and in the complexity of computation. As a simple example, we propose a general-purpose molecular system consisting of logical gates and sensors. As a more complex example, we present a new idea of constructing a DNA automaton by a chain reaction system, which can have an arbitrary number of states.

Polyphosphate:AMP phosphotransferase as a polyphosphate-dependent nucleoside monophosphate kinase in Acinetobacter johnsonii 210A.

We have cloned the gene for polyphosphate:AMP phosphotransferase (PAP), the enzyme that catalyzes phosphorylation of AMP to ADP at the expense of polyphosphate [poly(P)] in Acinetobacter johnsonii 210A. A genomic DNA library was constructed in Escherichia coli, and crude lysates of about 6,000 clones were screened for PAP activity. PAP activity was evaluated by measuring ATP produced by the coupled reactions of PAP and purified E. coli poly(P) kinases (PPKs). In this coupled reaction, PAP produces ADP from poly(P) and AMP, and the resulting ADP is converted to ATP by PPK. The isolated pap gene (1,428 bp) encodes a protein of 475 amino acids with a molecular mass of 55.8 kDa. The C-terminal region of PAP is highly homologous with PPK2 homologs isolated from Pseudomonas aeruginosa PAO1. Two putative phosphate-binding motifs (P-loops) were also identified. The purified PAP enzyme had not only strong PAP activity but also poly(P)-dependent nucleoside monophosphate kinase activity, by which it converted ribonucleoside monophosphates and deoxyribonucleoside monophosphates to ribonucleoside diphosphates and deoxyribonucleoside diphosphates, respectively. The activity for AMP was about 10 times greater than that for GMP and 770 and about 1,100 times greater than that for UMP and CMP.

LOCAL SEARCH BY CONCENTRATION-CONTROLLED DNA COMPUTING

Concentration-controlled DNA computing is presented for accomplishing a local search for the solution of a shortest path problem. In this method, the concentrations of DNA representing edges are determined according to the costs on edges, and then the hybridization process is performed. Since the concentrations of hopeless candidate solutions tend to be small after the hybridization process, a local search by concentration-controlled DNA computing is a promising approach. In order to discuss about the relationship between given costs on edges in the graph and concentrations of generated DNA paths, a simulation model of the hybridization process is used and the results of a laboratory experiment are shown.

Hairpin-based state machine and conformational addressing: Design and experiment

In this paper, we propose a new architecture for a multi-state DNA machine whose conformation of repeated hairpin structures changes sequentially in response to input oligomers. As an application of the machine, we also propose molecular memory in which the machine is used as a memory unit. Addressing in the memory is realized through state transitions of the machine. We then describe a method for designing DNA sequences of the machine, which exhaustively checks conformational changes of the machine by dividing its secondary structure into hairpin units. The method is based on the minimum free energy of the structure, the structure transition paths, and the total frequency of optimal and suboptimal structures. DNA sequences designed by the method were tested in a chemical experiment in which a machine consisting of two hairpins was actually constructed. As a result, we verified that the multi-state DNA machine realized the expected changes in its secondary structure.

Hierarchical DNA Memory Based on Nested PCR

This paper presents a hierarchical DNA memory based on nested PCR. Each DNA strand in memory consists of address blocks and a data block. In order to access specific data, we specify the order of the address primers, and nested PCR are performed by using these primers. Our laboratory experiments are also presented to demonstrate the feasibility of the proposed memory.

Conformational Addressing Using the Hairpin Structure of Single-Strand DNA

In this paper, we demonstrate through a chemistry experiment that conformational addressing can be achieved using the hairpin structure of a DNA molecule. The hairpin structure made by single-strand DNA (ssDNA) self-hybridization is made into the address part of conformational addressing, and it is assumed that the memory is read by opening this hairpin. The hairpin is continuously arranged in order to divide the address by class. Reading a sub-address requires an appropriate input oligomer to be added, when the preceding hairpin has been opened. We investigated, through the chemistry experiment, whether it would be possible to open the hairpin by the addition of an input oligomer into a solution that contained a hairpin-formed ssDNA.

Effects of surface states on control characteristics of nano-meter scale Schottky gates formed on GaAs

Abstract Effects of surface states on gate control characteristics of nano-meter scale Schottky gates formed on GaAs are investigated both theoretically and experimentally. Special sample structures are used. They are metal–insulator–semi-conductor structures having nano-meter scale Schottky dot arrays for capacitance–voltage ( C–V ) measurements and metal–semi-conductor field effect transistor structures having nano-meter scale grating Schottky gates for current–voltage ( I–V ) measurements. Measured C–V and I–V results are compared with results of theoretical calculation on a computer. The effects of surface states are found to be two-fold. Namely, it is shown that control characteristics of nano-meter scale Schottky gates are strongly degraded by the presence of Fermi level pinning caused by surface states on the free surface surrounding the gate. It is also shown that a significant amount of gate-induced lateral charging of surface states takes place around the gate periphery, effectively increasing the gate dimension. These results indicate the critical importance of control of surface states in nano-devices using nano-meter scale Schottky gates.