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Masayasu Kuwahara

Publications and source records attributed to Masayasu Kuwahara.

At least 19 recordsLinked to original sources
PubMed2006

Influence of cationic molecules on the hairpin to duplex equilibria of self-complementary DNA and RNA oligonucleotides.

A self-complementary nucleotide sequence can form both a unimolecular hairpin and a bimolecular duplex. In this study, the secondary structures of the self-complementary DNA and RNA oligonucleotides with different sequences and lengths were investigated under various solution conditions by gel electrophoresis, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy and a ultraviolet (UV) melting analysis. The DNA sequences tended to adopt a hairpin conformation at low cation concentrations, but a bimolecular duplex was preferentially formed at an elevated cationic strength. On the other hand, fully matched RNA sequences adopted a bimolecular duplex regardless of the cation concentration. The thermal melting experiments indicated a greater change in the melting temperature of the bimolecular duplexes (by approximately 20 degrees C) than that of the hairpin (by approximately 10 degrees C) by increasing the NaCl concentration from 10 mM to 1 M. Hairpin formations were also observed for the palindrome DNA sequences derived from Escherichia coli, but association of the complementary palindrome sequences was observed when spermine, one of the major cationic molecules in a cell, existed at the physiological concentration. The results indicate the role of cations for shifting the structural equilibrium toward a nucleotide assembly and implicate nucleotide structures in cells.

Cations↗
PubMed2006

Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.

We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

DNA↗
PubMed2006

Biomolecular sensor based on fluorescence-labeled aptamer.

Fluorescent DNA probes for L-argininamide were developed by a combination of DNA aptamers and fluorophore-quencher pairs. These molecules were synthesized by a combination of pre- and post-synthetic modification methods. The fluorescence-labeled aptamer could detect L-argininamide specifically. The binding affinities were defined by the binding affinity of the original aptamer to indicate that the end labeling of the aptamer did not influence the affinities.

Aptamers, Nucleotide↗
PubMed2006

Substrate property and incorporation accuracy of various dATP analogs during enzymatic polymerization using thermostable DNA polymerases.

DNA aptamers and DNAzymes with similar function to antibodies and enzymes can be produced by in vitro selection. They would be useful as research tools for molecular biology and as indicators of specific substances for the analysis of clinical and food samples. Furthermore, development of modified DNA molecules aimed to diversify function and improve activity has recently proceeded by introducing functionalities to these DNA molecules. Such functional modified DNA molecules with an aimed activity screened from a random sequence pool of modified DNA prepared by a polymerase reaction. To enhance potential of selection library and expand diversity of modified DNA that can be synthesized enzymatically, modified analogs of 2'-deoxyadenosine triphosphate were synthesized and their substrate properties for some thermostable DNA polymerases in polymerase chain reactions (PCR) were investigated. Modified DNAs were sequenced in order to analyze incorporation accuracy of modified dATP during PCR.

DNA-Directed DNA Polymerase↗
PubMed2006

Synthesis and property of DNA labeled with fluorescent acridone.

Triphosphate of a thymidine analogue bearing acridone was prepared from a modified thymidine nucleotide with a terminal amino group at C5 position and an acridone derivative as a new fluorecsent-tagged nucleotide. The acridone-tagged nucleotide was incorporated into DNA enzymatically during PCR using KOD Dash DNA polymerase. Further, we introduced the acridone derivative into an amino-modified DNA chemically by post-synthetic modification, and investigated their fluorescence properties and hybridization ability. The new fluorescent-labeled DNA bearing acridone will be useful as a DNA probe.

Acridines↗
PubMed2005

Direct PCR amplification of various modified DNAs having amino acids: convenient preparation of DNA libraries with high-potential activities for in vitro selection.

We synthesized modified 2'-deoxyuridine triphosphates bearing amino acids at the C5 position and investigated their substrate properties for KOD Dash DNA polymerase during polymerase chain reaction (PCR). PCR using C5-modified dUTP having an amino acyl group (arginyl, histidyl, lysyl, phenylalanyl, tryptophanyl, leucyl, prolyl, glutaminyl, seryl, O-benzyl seryl or threonyl group) gave the corresponding full-length PCR products in good yield. Although dUTP analogues bearing aspartyl, glutamyl or cysteinyl were found to be poor substrates for PCR catalyzed by KOD Dash DNA polymerase, optimization of the reaction conditions resulted in substantial generation of full-length product. In the case of reaction using dUTP analogue having a cysteinyl group, addition of a reducing agent improved the reaction yield. Thus, PCRs using KOD Dash DNA polymerase together with amino acyl dUTP provide convenient and efficient preparation of various modified DNA libraries with potential protein-like activities.

Amino Acids↗
PubMed2005

Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase.

Thymidine analogues bearing a variety of functional groups at the C5-position via an amino-linker arm were prepared and the substrate activity for PCR using thermophilic KOD Dash DNA polymerase was examined. The enzyme accepted the thymidine analogues bearing pyridine, imidazole, biotin, a cationic-charged guanidinium, a cationic-charged amino, mercaptopyridyl and phenanthrolne groups at the C5-position, forming the corresponding PCR product. However, a thymidine analogue bearing a carboxyl group at the C5-position was a poor substrate and the corresponding PCR products could not be obtained. The thymidine analogue bearing a mercapto group was also a poor substrate for the enzyme, because it dimerized by disulfide linkage under PCR conditions. The enzyme hardly accepts the thymidine analogues with a negatively-charged carboxyl group or a bulky group as a substrate. KOD Dash DNA polymerase, having a broader substrate specificity than any other DNA polymerase, will expand the variety of modified DNAs that can be prepared by PCR.

Amines↗
PubMed2005

Coformational switch of oligonucleotide induced by spermine.

Conformational change of nucleic acids induced by a small molecule is essential for many biological reactions and useful for biotechnology. To construct a spermine-responsive nucleic acid, we designed DNA sequences which can potentially form a unimolecular hairpin loop structure and a bimolecular duplex by itself. On the basis of UV melting and native PAGE experiments, we found four DNA oligomers of d16cg, d14cg, d12cg, and d14ta adopting different secondary structures depending on the spermine concentration. Their dissociation constants (Kd) with spermine were estimated in the order of 10(-6).

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

Comparison study on PCR amplification of modified DNA by using various kinds of polymerase and modified nucleoside triphosphates.

A series of C5 substituted analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate were synthesized and investigated their substrate properties for thermostable DNA polymerases during polymerase chain reaction (PCR). The PCR assays indicated that the relative yield of the full-length product is dependent on whether the substitution is on dU or dC, and on the nature of the polymerase.

DNA↗
PubMed2005

Screening of a glutamic acid-binding aptamer from arginine-modified DNA library.

A binder that is specific for a target will be applicable to detection of a variety of molecules such as bioactive substances, environmental contaminants and so on. Nucleic acid aptamers obtained by in vitro selection method are interesting and promising binders. To enhance the ability of nucleic acid aptamers, we prepared a combinatorial modified DNA library containing arginyl uracil base by using arginyl dUTP, then attempted screening of glutamic acid-binding aptamers from the library by in vitro selection method.

Aptamers, Peptide↗
PubMed2004

Synthesis and antisense properties of oligodeoxyribonucleotides containing C5-substituted arabinofuranosyluracil.

An oligodeoxyribonucleotide (ODN) containing three C5-substituted arabinofuranosyluracils was synthesized by the post-synthetic modification method from the ODN containing three C5-substituted 2,2'-anhydrouridines. The stability of the modified ODN/DNA duplex was lower than that of the corresponding normal duplex but that of the modified ODN/RNA duplex showed little change. The modified ODN could induce RNase H activity and was resistant against nuclease.

Arabinofuranosyluracil↗
PubMed2004

Sialyllactose-binding modified DNA aptamer bearing additional functionality by SELEX.

We produced a novel cationic-charged modified DNA aptamer for sialyllactose that is a ubiquitous component of the cell surface responsible for the infection of several viruses by using the magnetic-particle-based SELEX method. After 13 rounds of selection we selected 22 clones as sialyllactose-binding DNA aptamers composed of several modified thymidines. The DNA aptamers could form a three-way junction structure that likely forms a binding site for siallyllactose. The three-way junction structure contains several modified thymidines bearing a positively-charged amino group at the C5 position, which could enhance the binding ability for silalyllactose which has a negatively-charged carboxyl group. The dissociation constant of the aptamer that showed the strongest sialyllactose-binding ability among the clones of the aptamers was 4.9 microM.

Binding Sites↗
PubMed2004

Detection of biomolecule by aptamer beacon.

Labeled oligodeoxyribonucleotide bearing fluorescent dyes at both ends and aptamer sequence for adenosine 5'-monophosphate (AMP) was synthesized. Fluorescence spectra of labeled aptamer were not so much different between with and without AMP. This result suggests the binding of AMP didn't cause the global structural change to the aptamer. Therefore, we used short complementary DNA (SCD) as an assistant DNA, which is an unmodified 11mer and have a complementary sequence of 5'-region of the labeled aptamer. In the presence of SCD, the fluorescence intensities decrease with increasing the concentration of AMP compared with a change in absence of SCD.

Adenosine Monophosphate↗
PubMed2004

Screening of modified DNA aptamers that recognize DNA secondary structure.

We attempted to utilize in vitro selection techniques to develop modified DNA aptamers that are specific for DNA secondary structure including a single-mismatch base pair. For the selection, two kinds of random modified DNA library were prepared by an enzymatic method using modified 2'-deoxynucleoside 5'-triphosphates and KOD Dash DNA polymerase: one library consists of modified DNA including amino functionality and another consists of modified DNA including both hydroxypropynyl and guanidium functionalities.

Aptamers, Nucleotide↗
PubMed2003

Substrate properties of C5-substituted pyrimidine 2'-deoxynucleoside 5'-triphosphates for thermostable DNA polymerases during PCR.

In order to enhance a collection of modified deoxynucleoside triphosphates useful for in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) techniques, we designed and synthesized modified analogues of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate bearing a flexible and hydrophilic 7-amino-2,5-dioxaheptyl linker at a C5 position. Both analogues were found to be substrates for thermostable DNA polymerases which belong to an evolutional family B during PCR.

Base Sequence↗
PubMed2003

Convenient synthesis of oligodeoxyribonucleotides bearing arabinofuranosyl pyrimidine derivatives and its duplex formation with complementary DNA.

The oligodeoxyribonucleotides bearing 2,2'-anhydro-beta-D-arabinofuranosyluracil derivatives were synthesized and the modified residue was converted to beta-D-arabinofuranosyluracil derivatives or beta-D-arabinofuranosylisocytosine derivatives by post-synthetic modification method. The melting profiles of their ODNs with complementary DNA were studied.

Arabinose↗