Biomedical subjects

Hirotaka Miyachi

Publications and source records attributed to Hirotaka Miyachi.

4 recordsLinked to original sources

PubMed2006

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance.

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.

Actins↗

PubMed2005

Exo-Taq-based detection of DNA-binding protein for homogeneous and microarray format.

The study of DNA-protein interactions is of great importance to understand basic cellular processes such as transcription, replication and recombination. In this research, we developed a novel detection system for DNA-binding proteins (DBPs) involving the exonuclease (Exo) III and Taq DNA polymerase reactions. The system consists of three steps, as follows: the target DBP in the sample solution is incubated with probe DNA, and the probe is digested with Exo III and then extended with Taq using fluorescent dye-labeled dUTP as a substrate. The DBP protects the probe from digestion by Exo III. Therefore, only the DBP-bound probe allows the following extension. We examined this system using the lambda phage Cro repressor in a homogeneous format. The fluorescence image after gel electrophoresis showed a specific band. We also found that this system could be applied to the rapid and efficient detection of DBPs in stem and loop ds-DNA array formats. These results suggest that our method is useful as a new tool for analyzing DNA-protein interactions.

Base Sequence↗

PubMed2004

Imprinted polymer layer for recognizing double-stranded DNA.

A method of preparing a thin polymer layer able to recognize double-stranded DNA (dsDNA) was developed by using 2-vinyl-4,6-diamino-1,3,5-triazine (VDAT) as a functional monomer for creating a DNA-imprinted polymer. The formation of hydrogen bonds between VDAT and A-T base pairs in dsDNA was confirmed by measuring the effects of VDAT on the melting point and the NMR and CD spectra of dsDNA. An imprinted polymer that can recognize dsDNA of the verotoxin gene was prepared by polymerizing VDAT, acrylamide, a crosslinking agent, and the template verotoxin dsDNA on a silanized glass surface. The specificity of this polymer layer for binding verotoxin dsDNA was investigated by using fluorescent-labelled dsDNAs. The fluorescence intensity of the polymer layer after binding verotoxin dsDNA was twice as high as after binding oligo(dG)-oligo(dC), indicating that verotoxin dsDNA was preferentially bound to the polymer imprinted with verotoxin dsDNA. The kinetics of verotoxin dsDNA binding to the imprinted polymer were analyzed by surface plasmon resonance measurements. The dissociation constant (KD) was low, of the order of 10(-9)M.

Acrylic Resins↗

PubMed2004

Single nucleotide polymorphism typing on DNA array with hydrophobic surface fabricated by plasma-polymerization technique.

A DNA array has been fabricated on glass substrates, which enables high-throughput analysis of single-base mismatches. In this work, microfabrication-compatible plasma-polymerization (PP) method was used for immobilizing probe DNAs to study the hybridization behavior by changing surface properties. The immobilization matrix consisting of 35 A of PP layer, applied additionally on the streptavidin absorbed hexamethyldisiloxane (HMDS)-PP layer, was constructed on the substrates to anchor biotinylated DNA probes onto the surface. The hydrophobic immobilization matrix was considered to enhance hybridization accuracy and efficiency, compared with its hydrophilic acetonitrile-PP layers. The oligonucleotide arrays fabricated on HMDS-PP surface were shown to be effective in detection of single nucleotide polymorphisms (SNPs) of ApoE gene.

Apolipoproteins E↗