Shokichi Ohuchi

Controlled Microwave Heating Accelerates Rolling Circle Amplification

Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.

Effect of Microwaves on DNA and Proteins

The Rolling Circle Amplification (RCA) is an enzymatic synthesis method of deoxyribonucleic acid (DNA) strands with repeated sequence of a circulate template-DNA. Microwave heating technology is applied to organic and inorganic chemistry to produce useful effects such as rapid heating, selective heating, decreased reaction times, and improved product yield. However, the mechanisms underlying the thermal or non-thermal effects, i.e., reaction-promoting effects, of MW chemistry are still unclear. We focus on the molecular perspective to investigate the effect of MWs via a DNA amplification reaction that utilizes enzymes. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. We show that microwave heating facilitated the synthesis of repetitive DNA through RCA using the four DNA polymerases. Analysis of the temperature profiles of each RCA component subjected to microwave heating revealed the selectivity heating of buffer components compared with primers, template DNA, dNTP, and RNase-free water. Further, we showed the relation between the microwave power loss and RCA components via dielectric measurements, cavity resonator feature measurement, and electromagnetic simulation. The buffer containing ions of the RCA components was selectively heated via microwave irradiation in the TM010 cavity resonator.

Correlation of microwave power dependence and temperature dependence on enzymatic reaction under microwave irradiation

Summary form only given. Chemistry under microwave irradiation shows specific phenomena to overturn common sense. On the other hand, these phenomena also have resulted in time shortening effect for a number of chemical processes. These effects have been led to the breakthrough innovation. Most of their specific phenomena are chemical reactions. However, the mechanisms at the molecular level, there is still a lot of discussion. As research aimed at elucidating the mechanism, we have been working on organic reactions, enzymatic reactions, and microbial cultivation. In this study, we proceeded to study about the enzymatic hydrolysis reaction of invertase and lipase as a model reaction. Invertase, keeping the reaction temperature constant, was experimentally giving various microwave power. Lipase, for reaction substrates of different polarity, examined the effect of microwave power. In order to precisely control the temperature and the microwave output, the reaction vessel was fitted with a cooling jacket. For the obtained results, physical-chemical analysis such as the Michaelis-Menten equation and the Arrhenius equation has been carried out. First, the results of invertase, the phenomenon of “microwave output dependent” was found. It is speculated that corresponds to the “temperature dependence” of an enzyme reaction. This phenomenon has been found already also in the microbial cultivation. Next, the results of lipase, correlation between the substrates of a polar and enzyme kinetics was observed. Molecular motion is controlled in rotational motion by the electromagnetic energy, it is estimated to the formation of the specific heating mechanism.

Microwave assisted trypsin digestion with cavity type resonator reactor as an innovative proteomics technology

Sumamry form only given. The word “omics” indicates the study of a total information in biological cell, such as the genome, which is all DNA in a living cell, or the proteome which is all the proteins. Omics technologies of genomics and proteomics, such as tailor-made treatment, it has been positioned in the center of the future of medical technology. The efficiency of this technology is also expected by microwave technology. Microwave technology, for the time shortening of omics pretreatment reaction, has been fully achieved. The corresponding to the small-scale is also strongly desired to high-throughput technology such as a well plate. The corresponding to the small-scale is strongly desired to high-throughput technology such as a well plate. In this study, we tried the microwave assisted proteomics for enzymatic hydrolysis of protein molecules with a protease tripsin. Chicken egg white-derived lysozyme having a molecular weight 14,300 as a model protein was used in the enzymatic hydrolysis. The molar ratio of trypsin and lysozyme was adjusted to 1 : 50, reductive alkylation lysozyme was digested with 37°C, and analyzed by MALDI-TOF MS after the reaction. An irradiation reactor of cavity resonator type was applied to enzyme reaction microwaves continuously irradiated with microwaves of 16 W, and maintained at 37°C, of the reaction temperature. The conventional method was used with a water bath. As a result, reaction time was reduced from 20 hours to 10 minutes. It was found that the reaction had proceeded completely, from Mass spectrum. In the enzymatic hydrolysis, there is the problems to digest for oneself. However, the autolysis of trypsin under microwave irradiation was not detected. Next, we had checked by MASCOT searching after TOFMS analysis, it was confirmed which part of the protein is cleaved from the protein fragments. Enzyme-substrate concentration, time of 1 to 10, the sequence coverage of 84% by microwave irradiation was 57% under normal conditions. Trypsin recognized amino acid sequence, which means that 84% is broken, it was considered to cleave amino acid residues inside the protein.