Shun-ichi Matsuura

Single-molecule PCR using water-in-oil emulsion.

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.

Indirect micromanipulation of single molecules in water-in-oil emulsion.

Based on real‐time observation and micromanipulation, analytical methods for single DNA molecules have been under development for some time. Precise manipulation, however, is still difficult because single molecules are too small for conventional techniques. We have developed a chemical reaction system that uses water droplets in oil as containers of materials. The water droplets can be manipulated by optical force. The manipulation of the water droplets permits the fusion of two selected droplets. This process corresponds to mixing of different samples. We designate this system as “w/o (water‐in‐oil emulsion) microreactor system”, and each droplet can be thought of as a “microreactor”. In this system, single molecules can be manipulated readily, as a molecule can be contained in a μm‐sized microreactor. The microreactor utilizes extremely small quantities of samples, therefore, reactions are rapid, as diffusion times in the microreactor are very short. The manipulation technique of the microreactors based on optical force has been applied to induce fusion between microreactors loaded with DNA and YOYO, a fluorescent dye that binds to DNA. This fusion induced a rapid binding of YOYO.

Enhancement in thermal stability and resistance to denaturants of lipase encapsulated in mesoporous silica with alkyltrimethylammonium (CTAB)

We assembled a highly durable conjugate with both a high-density accumulation and a regular array of lipase, by encapsulating it in mesoporous silica (FSM) with alkyltrimethylammonium (CTAB) chains on the surface. The activity for hydrolyzing esters of the lipase immobilized in mesoporous silica was linearly related to the concentration of lipase, whereas that of non-immobilized lipase showed saturation due to self-aggregation at a high concentration. The lipase conjugate also had increased resistance to heating when stayed in the silica coupling with CTAB. In addition, encapsulating the enzyme with FSM coupled CTAB caused the lipase to remain stable even in the presence of urea and trypsin, suggesting that the encapsulation prevented dissociation and denaturing. This conjugate had much higher activity and much higher stability for hydrolyzing esters when compared to the native lipase. These results show that FSM provides suitable support for the immobilization and dispersion of proteins in mesopores with disintegration of the aggregates.

A new DNA combing method for biochemical analysis

A simple molecular combing method for analysis of biochemical reactions, called the moving droplet method, has been developed. In this method, small droplets containing DNA molecules run down a sloped glass substrate, and this creates a moving interface among the air, droplet, and substrate that stretches the DNA molecules. This method requires a much smaller volume of sample solution than other established combing methods, allowing wider application in various fields. Using this method, lambdaDNA molecules were stretched and absorbed to a glass substrate, and single-molecule analysis of DNA synthesis by DNA polymerases was performed.

An enzyme-encapsulated microreactor for efficient theanine synthesis

A flow-type microreactor containing glutaminase-mesoporous silica composites with 10.6 nm pore diameter (TMPS10.6) was developed for the continuous synthesis of theanine, a unique amino acid. High enzymatic activity was exhibited by the local control of the reaction temperature.

Synthesis of amino acid using a flow-type microreactor containing enzyme–mesoporous silica microsphere composites

A flow-type microreactor containing composite materials of a theanine synthetase (glutaminase) and mesoporous silica with 23.6 nm pore diameter (SBA-15 microsphere) was developed for the continuous synthesis of L-theanine, a unique amino acid. Enzyme-immobilisation ability and enzymatic activity in the SBA-15 microsphere with large mesopores were higher than those of SBA-15 with a 5.4 nm pore diameter. Moreover, the glutaminase–SBA-15 microsphere composites displayed higher selectivity in theanine production than the free enzyme did in a batch experiment. A direct visualization of composites of fluorescently labelled glutaminase and SBA-15 microsphere immobilised in the flow channel of the microreactor by a combination of differential interference contrast and fluorescence microscopy revealed that the enzymes were uniformly dispersed throughout the mesoporous silica particles, because of the successful encapsulation of the enzyme. The enzyme-encapsulated microreactor exhibited a high conversion of L-glutamine to L-theanine with local control of the reaction temperature. In addition to this advantage of the microreaction system, the microreactor enabled the on-off regulation of enzymatic activity during continuous theanine synthesis by controlling the reaction temperature or the pH of the substrate solution.

Activation of restriction enzyme by electrochemically released magnesium ion

Observation and cutting of DNA molecules at intended positions permit several new experimental methods that are completely different from conventional molecular biology methods; therefore several cutting methods have been proposed and studied. In this paper, a new cutting method for a DNA molecule by localizing the activity of a restriction enzyme is presented. Since most restriction enzymes require magnesium ions for their activation, local restriction enzyme activity can be controlled by the local concentration of magnesium ions. Applying a direct current (dc) voltage to a needle electrode of metallic magnesium made it possible to control the local magnesium ion concentration at the tip of the needle. The restriction enzyme was activated only when magnesium ions were electrochemically supplied.

Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State

Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA.

Nanoporous scaffold for DNA polymerase: pore-size optimisation of mesoporous silica for DNA amplification

Composites of thermostable DNA polymerase and mesoporous silicas with different pore diameters were used for DNA amplification. The enzyme was selectively immobilised on the mesopores, and DNA amplification activity was retained by regulating the pore size of the mesoporous silicas.

Enzyme Immobilization in Mesoporous Silica for Enhancement of Thermostability

Direct enzyme immobilization by encapsulation in the pores of mesoporous silica particles enhances protein thermal and chemical stability. In this study, we investigated the effect of pore size on the thermostability and catalytic activity of Escherichia coli glutaminase YbaS encapsulated under high temperature conditions in two SBA-type mesoporous silicas: SBA5.4 and SBA10.6 with pore diameters of 5.4 and 10.6 nm, respectively. The changes in enzyme conformation under high temperature conditions were assessed using PSA, a benzophenoxazine-based fluorescent dye that is sensitive to denatured aggregated proteins. The results showed that YbaS adsorption to SBA10.6 was higher than that to SBA5.4 and that SBA10.6-encapsulated YbaS was more resistant to heat treatment and maintained higher conformational stability than SBA5.4-encapsulated or free enzyme. Moreover, the heat-treated YbaS-SBA10.6 composite demonstrated high catalytic activity in glutamine hydrolysis. Thus, enzyme encapsulation in suitable silica mesopores can prevent heat-induced denaturation and subsequent aggregation of the enzyme.