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Biomedical subjects

Takahiko Nojima

Publications and source records attributed to Takahiko Nojima.

At least 19 recordsLinked to original sources
PubMed2007

Direct modification of mRNA by ferrocenyl carbodiimide and its application to electrochemical detection of mRNA.

Ferrocenyl carbodiimide (1) could be used for the direct labeling of synthetic RNA and expressed mRNA in vitro with the electrochemically active ferrocene moieties. These RNAs modified by 1 could be detected electrochemically coupled with a DNA probe-immobilized electrode. After hybridization of 1.1 Kb mRNA modified by 1 with the DNA probe-immobilized electrode, the peak charge observed by an Osteryoung square wave voltammetry (SWV) measurement correlated well with the concentration of mRNA, having a detection limit at the sub nanogram level.

Carbodiimides↗
PubMed2006

G quadruplex-based FRET probes with the thrombin-binding aptamer (TBA) sequence designed for the efficient fluorometric detection of the potassium ion.

The dual-labeled oligonucleotide derivative, FAT-0, carrying 6- carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) labels at the 5' and 3' termini of the thrombin-binding aptamer (TBA) sequence 5'-GGT TGG TGT GGT TGG-3', and its derivatives, FAT-n (n=3, 5, and 7) with a spacer at the 5'-end of a TBA sequence of T(m)A (m=2, 4, and 6) have been designed and synthesized. These fluorescent probes were developed for monitoring K(+) concentrations in living organisms. Circular dichroism, UV-visible absorption, and fluorescence studies revealed that all FAT-n probes could form intramolecular tetraplex structures after binding K(+). Fluorescence resonance energy transfer and quenching results are discussed taking into account dye-dye contact interactions. The relationship between the fluorescence behavior of the probes and the spacer length in FAT-n was studied in detail and is discussed.

Aptamers, Nucleotide↗
PubMed2006

Electrochemical detection of DNase I activity.

DNase I in one microl of the water could quantitate electrochemically with the detection limit of 0.01 units (ca. 20 pg) by using the ferrocenyl oligonucleotide-immobilized electrode prepared by thiolated oligonucleotide and ferrocenyl carbodiimide as a simple labeling reagent of redox unit.

Carbodiimides↗
PubMed2006

Electrochemical SNP detection.

Detection of mismatched base on a DNA duplex, which is important for the search of the heterozygote, achieved by the comparison with the reactivity of ferrocenylcarbodiimide (1) developed by our group. Mismatched thymine base on the 30-meric DNA duplex as a model of SNPs in lipoprotein lipase (LPL) gene, could react with 1 in 20 mM borate buffer (pH 8.5) and 0.1 M NaCl containing 20% DMSO at 37 degrees C. DNA duplex modified by 1 in the mismatched thymine bases could be detected by its shifted migration time in microTAS electrophoresis.

Base Pair Mismatch↗
PubMed2006

Controlling the expression ratio of two proteins by inserting a terminator between the two genes.

An approach was developed in controlling the expression ratio of two genes encoded in DNA by inserting a terminator sequence between the two genes. Escherichia coli ribosomal RNA operon T1 terminator, or E. coli tryptophane attenuater was introduced between two green fluorescent protein (GFP) mutants: ultraviolet-optimized mutant (GFPuv: F99S, M153T, V163A) and blue-shifted mutant (BFP: F64L, S65T, T145F). The ratio of the fluorescence intensity of BFP to GFPuv after transcription and translation reflected the termination efficiency of the transcriptional termination activity of the inserted terminator.

Escherichia coli↗
PubMed2006

Preparation of carbodiimide-terminated dithiolane self-assembly monolayers as a new DNA-immobilization method.

A carbodiimide derivative having a dithiolane part at its terminus was designed and synthesized for use to construct carbodiimide-coated self-assembly monolayers (SAMs) on a gold surface with 6-mercaptohexanol (6MH). When treated with poly(dT), poly(dA), or poly(dA)poly(dT), only poly(dT) was immobilized on the surface of the SAMs through a specific reaction of the free imino moiety of thymine (T) with the carbodiimide moiety. The carbodiimide-covered SAM treated with probe DNA was tested in hybridization with sample DNA. Its hybridization efficiency was estimated by ferrocenylnaphthalene diimide (FND), described previously and the result revealed that the carbodiimide-covered SAM electrode can immobilize a DNA probe through the thymine moiety not involved in base pairing. The resulting electrode was capable of hybridizing with the target DNA, as proven by an increased current response of FND.

Carbodiimides↗
PubMed2005

Immobilization of RNase S-Peptide on a single-stranded DNA-fixed gold surface and effective masking of its surface by an acridinyl poly(ethylene glycol).

Oligonucleotide-peptide conjugate was synthesized by coupling of RNase S-peptide to a 24-mer single-stranded DNA (ssDNA) oligonucleotide to be immobilized on its complementary ssDNA oligonucleotide-fixed gold surface of sensor chip or electrode. Immobilization of on the ssDNA-fixed gold surface through DNA duplex formation was confirmed by quartz crystal microbalance (QCM) and electrochemical measurements. After treating with a synthetic acridinyl poly(ethylene glycol) (APEG), specific interaction of S-protein with the S-peptide immobilized on the gold surface was demonstrated by QCM without nonspecific adsorption of unrelated proteins such as BSA and RNase A at the surfaces. This result suggested that the acridine parts of APEG could bind to the DNA duplex on the gold surface and the poly(ethylene glycol) parts were fastened on the surface to resist the adsorption of proteins. Thus, the combination of oligonucleotide-peptide conjugate, ssDNA-fixed chip and APEG with effective masking property provides a new tool for the analysis of specific peptide-protein interactions without disturbance by other unrelated proteins.

Acridines↗
PubMed2005

Electrochemical telomerase assay with ferrocenylnaphthalene diimide as a tetraplex DNA-specific binder.

Spectroscopic studies revealed that ferrocenylnaphthalene diimide (1) can bind to tetraplex DNA at high potassium ion concentration. The tetraplex DNA was stabilized by the binding of 1, and this effect was larger than that of any other tetraplex stabilizers, which are known as a telomerase inhibitor. Quantitative analysis with circular dichroism and a quartz crystal microbalance strongly suggested a 3:1 binding stoichiometry of 1 to the tetraplex DNA. The telomere sequence could be extended by telomerase with the telomerase substrate primer on the surface of an electrode as proven by an increased current signal of 1 bound to the tetraplex DNA formed on the electrode. This is the first example of electrochemical detection of telomerase activity without relying on PCR.

Circular Dichroism↗
PubMed2005

Investigation of ferrocenyl carbodiimide (FCDI) in the modification reaction of nucleic acids.

Ferrocenyl carbodiimide (FCDI) was allowed to react with thymine or guanine bases of single stranded DNA in borate buffer (pH 9.5) at 37 degrees C for a short period of time. The reactivity of FCDI with the guanine bases was smaller than that with the thymine ones and the prolongation of the reaction time resulted in a lower yield. The reactivity for double stranded oligonucleotide was extremely decreased from that for the single stranded one. One base mismatched double stranded oligonucleotides could react with FCDI, albeit slowly. These results suggested that the reactivity of FCDI was variable depending on the kind of bases and the oligonucleotide structure.

Base Pair Mismatch↗
PubMed2005

Complexation of thrombin-binding aptamer oligonucleotide carrying fluorescence resonance energy transfer (FRET) dyes at both termini with potassium ion.

Oligonucleotide derivatives, F-TBA-T and F-7TBA-T carrying thrombin-binding aptamer (TBA) sequence and FAM and TAMRA dyes at their each end were designed to achieve the fluorometric monitoring of potassium ion (K+) concentrations in a living organisms. These oligonucleotides could form a tetraplex structure upon addition of K+. Spectrophotometric experiments revealed that the fluorescence enhancement on the fluorescence resonance energy transfer (FRET) was larger in the case of F-7TBA-T having 7-nucleotides at 5'-end of TBA sequence than in the case of F-TBA-T due to the elimination of the quenching by dye-dye interaction.

Aptamers, Nucleotide↗
PubMed2005

Electrochemical detection of telomeric quadruplex DNA using ferrocenyl naphthalene diimide.

Ferrocenylnaphthalene diimide (FND) could strongly bind to the synthetic telomere DNA, 5'-CAT GGT GGT TTG GGT TAG GGT TAG GGT TAG GGT TAC CAC-3', at 0.1 M KCl. The binding affinity of FND for the telomere DNA was ten times higher than that for calf thymus DNA under the conditions employed. Circular dichroism spectra suggested that the telomere DNA can form a tetraplex structure and FND can bind to it. Oligonucleotide extended by telomerase on the electrode could also be detected electrochemically by using FND, thereby suggesting the possibility that telomerase activity may be detected electrochemically.

DNA↗
PubMed2005

Genotyping of the human lipoprotein lipase gene by ferrocenylnaphthalene diimide-based electrochemical hybridization assay.

A ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay (FND-EHA) was applied to the detection of two mutations in human lipoprotein lipase (LPL) gene, G188E (one base transition) and Arita (one base deletion). A probe oligodeoxyribonucleotide of 13 bases representing the wild type (WT) sequence of LPL was immobilized on a gold electrode, followed by hybridization with a sample PCR product of 350 base pairs under conditions in which both WT and mutated (MT) sequences could form a duplex with the probe. The hybridized electrodes were soaked in an electrolyte containing FND under conditions in which only the mismatched duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex remaining on the electrode to give rise to a current signal. Blind tests were run to judge the genotype (WT/WT, WT/MT, or MT/MT) of 10 samples each for the G188E and Arita mutations and then, 8 and 10 of them were judged correctly, respectively.

Base Sequence↗
PubMed2005

Determination of the termination efficiency of the transcription terminator using different fluorescent profiles in green fluorescent protein mutants.

An approach in determining the intrinsic termination efficiency (%T) of transcription termination using green fluorescent protein (GFP) mutants was developed. This approach utilizes a cassette vector in which the tested terminator is introduced between two GFP mutant genes: an ultraviolet-optimized mutant (GFPuv: F99S, M153T, V163A) and a blue-shifted mutant (BFP: F64L, S65T, T145F). The ratio of the fluorescence intensity of BFP to GFPuv after transcription and translation represents the termination efficiency of the terminator. E. coli ribosomal RNA operon T1 terminator, phage lambda terminator site R2, E. coli tryptophane attenuater were introduced into the vector, and their transcriptional efficiencies were estimated as 89, 79, and 24%, respectively, showing good agreement with published data.

Base Sequence↗
PubMed2005

Interaction analysis of the carcinoembryonic antigen (CEA) with its monoclonal antibody immobilized on a gold surface using Fourier transform infrared reflection-absorption spectroscopy (FT-IR RAS).

A monoclonal antibody for the carcinoembryonic antigen (CEA) was immobilized on a gold chip surface covered by a self-assembled monolayer of 11-mercaptoundecanoic acid. Upon the addition of CEA, a Fourier transform infrared reflection-absorption spectroscopy (FT-IR RAS) measurement showed an increased absorption at around 1500 - 1700 cm(-1), corresponding to its amide structures. Another addition of CEA polyclonal antibody on this chip caused a further increase of the absorption in this region only after a treatment with CEA. This result shows that an antibody-fixed gold surface coupled with an FT-IR RAS measurement provides a new tool for detecting the antibody-antigen interaction.

Antibodies, Monoclonal↗