Hirofumi Kurita

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.

One-end immobilization of individual DNA molecules on a functional hydrophobic glass surface.

Abstract We demonstrate an effective method for DNA immobilization on a hydrophobic glass surface. The new DNA immobilizing technique is extremely simple compared with conventional techniques that require heterobifunctional crosslinking reagent between DNA and substrate surface that are both modified chemically. In the first process, a coverslip was treated with dichlorodimethylsilane resulting in hydrophobic surface. γ DNA molecules were ligated with 3′-terminus disulfide-modified 14 mer oligonucleotides at one cohesive end. After reduction of the disulfide to sulfhydryl (thiol) groups the resulting thiol-modified γ DNA molecules were reacted on silanized coverslip. Fluorescent observation showed that the thiol-modified γ DNA molecules were anchored specifically to the hydrophobic surface at one terminus, although non-specific binding of the DNA molecules was suppressed. It was observed that the one-end-attached DNA molecule was bound firmly to the surface and stretched reversibly in one direction when a d.c. electric field was applied.

Single-molecule measurement of strand breaks on large DNA induced by atmospheric pressure plasma jet

We report a single-molecule-based analysis of strand breakages on large DNA molecules induced by an atmospheric pressure plasma jet. We exposed DNA solution to an argon plasma jet; single-molecule observation that involved molecular combing was then used to measure the length of individual DNA molecules. The measuredDNA length showed that plasma exposure caused a marked change in length of DNA molecules. The rate of plasma-induced strand breakage on large random-coiled DNA molecules was determined using a simple mathematical model. For strand breaks on large DNA molecules the rate was estimated.

The Effect of Physical Form of DNA on ExonucleaseIII Activity Revealed by Single-molecule Observations

Single-molecule studies have revealed molecular behaviors usually hidden in the ensemble and time averaging of bulk experiments. Single-molecule measurement that can control physical form of individual DNA molecules is a powerful method to obtain new knowledge about correlation between DNA-tension and enzyme activity. Here we study the effect of physical form of DNA on exonucleaseIII (ExoIII) reaction. ExoIII has a double-stranded DNA specific 3′→5′ exonuclease activity and the digestion is distributive. We observed the ExoIII digestion of individual stretched DNA molecules from the free ends. The sequentially captured photographs demonstrated that the digested DNA molecule linearly shortened with the reaction time. We also carried out the single-molecule observation under random coiled form by pausing the buffer flow. The digestion rates obtained from both single-molecule experiments showed that the digestion rate under the stretched condition was two times higher than the random coiled condition. The correlation between physical form of DNA and digestion rate of ExoIII was clearly demonstrated by single-molecule observations.

Stretching of long DNA molecules in the microvortex induced by laser and ac electric field

A microvortex is generated around an infrared laser focus where an intense ac electric field is applied. The authors used this optoelectrostatic microvortex for stretching individual long DNAs. When λ-or T4-phage DNA molecules were introduced into the optoelectrostatic microvortex, they were stretched around the laser focus. In addition, especially for longer T4 DNA molecules, it was possible to keep it in stretching form for more than 30s. Using this method, length of DNA molecules can be measured without fixing to a substrate. This method can be applied to DNA molecules longer than about 10μm.

Analysis of the Inactivation Mechanism of Bacteriophage

Virus inactivation methods using atmospheric pressure discharge plasma have been actively studied these days. However, the predominant factor of the inactivation is not yet well understood. We believe that bacteriophages, which consist of only two components of nucleic acids and coat proteins, are suitable for the study of the inactivation mechanism because their simple structure can enable simple and clear analysis of the mechanisms. In this paper, inactivation of bacteriophage φX174, having some characteristics similar to influenza virus, was investigated using dielectric barrier discharge or corona discharge. In this experimental system using bacteriophage φX174, by transfection, deoxyribonucleic acid (DNA) activity can be separately analyzed from other factors such as coat proteins since it is possible to extract the nucleic acids from the bacteriophage. The coat proteins of the bacteriophage φX174 can be separately analyzed from its DNA as well. Plasma inactivation experiments were made in both wet and dry conditions. As a result, damage was found on both the DNA and the coat proteins of bacteriophage φX174 when it was exposed to the plasma. It was found that the damages given to the coat proteins were more predominant compared with the damages on the DNA.

\varphi\hbox{X}174

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.

by Atmospheric Pressure Discharge Plasma

Nonthermal atmospheric pressure plasma (NTP) has been extensively studied for biological and medical applications in recent years. Chemically active species are generated and injected into aqueous media subjected to plasma exposure. They play an important role in the activation and/or inactivation of biomolecules, such as nucleic acids, lipid, and proteins, in aqueous media. To evaluate the effect of NTP on these biomolecules at the molecular level, we have been considering large DNA molecules to be used as a biomarker. The single-molecule-based analysis of strand breakages induced by NTP, namely, the atmospheric pressure plasma jet (APPJ), on large DNA molecules has been performed. We demonstrated the facile estimation of the rate of double-strand breaks induced by the APPJ. Using this analysis, we have evaluated both the intensity of radical reaction and the protective effect on the reaction by adding antioxidant agents.

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

A high Tc SQUID system for biological molecules (DNA) detection is developed. This system is based on a hybridization process. Two strands in a DNA molecule are held together by hydrogen bonds between base pairs like a ladder. The two strands are referred to as being complementary each other. HPVB 33 (Human Papillomavirus Probes 33) was prepared as a DNA. One strand (Sample DNA) was labeled with Fe/sub 3/O/sub 4/ ultra-small magnetic particles and the other (probe DNA) was anchored on a glass slide. Then they were hybridized each other on the slide. After washing the excess sample DNA, the hybridized DNA was evaluated in the presence of excitation ac field by high Tc SQUID. The signal was initially proportional to the concentration of the sample DNA and then saturated. It means that the hybridization occurred successfully between the sample DNA and the probe DNA.

Radical reaction in aqueous media injected by atmospheric pressure plasma jet and protective effect of antioxidant reagents evaluated by single-molecule DNA measurement

Inactivation technology of viruses by atmospheric pressure discharge plasma has been studied actively. However, the predominant factor in the inactivation is still unclear. Bacteriophages, consisting of only nucleic acids and coat proteins, are suitable for the study of the inactivation mechanism due to their simple structure. In this study, inactivation of bacteriophage φX174, which is resistant to drying stress and has some characteristics similar to influenza virus, was investigated using dielectric barrier discharge (DBD) or corona discharge. We exposed wet or dry bacteriophage φX174 to discharge plasma. It is possible to extract the nucleic acids from the bacteriophage and to assay its plaque forming activity by transfection. Therefore, the damage given to the DNA can be separately analyzed. The coat proteins were also analyzed. Both DNA and coat proteins of plasma-treated φX174 phage were damaged, however, the damages of coat proteins were more significant compared to the DNA damages.