Yuuya Kasahara
Osaka University
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Featured researches published by Yuuya Kasahara.
Journal of the American Chemical Society | 2013
Yuri Imaizumi; Yuuya Kasahara; Hiroto Fujita; Shunsuke Kitadume; Hiroaki Ozaki; Tamaki Endoh; Masayasu Kuwahara; Naoki Sugimoto
Nucleic acid aptamers are receptors of single-stranded oligonucleotides that specifically bind to their targets. Significant interest is currently focused on development of small molecule aptamers owing to their applications in biosensing, diagnostics, and therapeutics involving low molecular weight biomarkers and drugs. Despite great potential for their diverse applications, relatively few aptamers that bind to small molecules have been reported, and methodologies to enhance and broaden their functions by expanding chemical repertories have barely been examined. Here we describe construction of a modified DNA library that includes (E)-5-(2-(N-(2-(N(6)-adeninyl)ethyl))carbamylvinyl)-uracil bases and discovery of high-affinity camptothecin-binding DNA aptamers using a systematic evolution of ligands by the exponential enrichment method. Our results are the first to demonstrate the superior efficacy of base modification on affinity enhancement and the usefulness of unnatural nucleic acid libraries for development of small molecule aptamers.
Molecules | 2010
Masayasu Kuwahara; Yuuki Takano; Yuuya Kasahara; Hiroki Nara; Hiroaki Ozaki; Hiroaki Sawai; Akio Sugiyama; Satoshi Obika
Recently, KOD and its related DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nucleic acids, but also sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA) which would be promising candidates for nucleic acid drugs. However, thus far, reasons for the effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated. Therefore, using mutated KOD DNA polymerases, we studied here their catalytic properties upon enzymatic incorporation of nucleotide analogues with base/sugar modifications. Experimental data indicate that their characteristic kinetic properties enabled incorporation of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2′-ONHCH2CH2-4′ linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effectiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demonstrated.
Analytical Chemistry | 2014
Yuka Kataoka; Hiroto Fujita; Yuuya Kasahara; Toshitada Yoshihara; Seiji Tobita; Masayasu Kuwahara
We newly synthesized thioflavin T (ThT) analogs for which the methyl group at the N3 position on the benzothiazole ring was replaced with either a ((p-(dimethylamino)benzoyl)oxy)ethyl group (ThT-DB) or a hydroxyethyl group (ThT-HE). In several neutral buffers, ThT-HE bound to a parallel guanine-quadruplex (G4) DNA and selectively emitted strong fluorescence at 74- to 240-fold higher intensities than those in the presence of double-stranded DNA (dsDNA), whereas ThT resulted in only 13- to 25-fold higher intensities. Furthermore, circular dichroism (CD) analyses using ThT, ThT-DB, and ThT-HE showed that these compounds could induce topological changes in G4. In addition, the different chemical structures of the N3 substituents could alter a G4-DNA conformation. These results indicate a great potential for N3-substituted ThT analogs as G4 probes and drug leads to achieve gene expression regulation.
Pharmaceuticals | 2013
Hiroto Fujita; Yuri Imaizumi; Yuuya Kasahara; Shunsuke Kitadume; Hiroaki Ozaki; Masayasu Kuwahara; Naoki Sugimoto
We recently selected DNA aptamers that bind to camptothecin (CPT) and CPT derivatives from a 70-mer oligodeoxyribonucleotide (ODN) library using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method. The target-binding activity of the obtained 70-mer CPT-binding DNA aptamer, termed CA-70, which contains a 16-mer guanine (G)-core motif (G3TG3TG3T2G3) that forms a three-tiered G-quadruplex, was determined using fluorescence titration. In this study, truncated fragments of CA-70 that all have the G-core motif, CA-40, -20, -19, -18A, -18B, -17, and -16, were carefully analyzed. We found that CA-40 retained the target-binding activity, whereas CA-20, -19, and -18B exhibited little or no binding activities. Further, not only CA-18A but also the shorter length fragments CA-17 and -16 clearly retained the binding activity, indicating that tail strands of the G-quadruplex structure can significantly affect the target binding of G-quadruplex DNA aptamers. Further analyses using circular dichroism (CD) spectroscopy and fluorescence polarization (FP) assay were conducted to investigate the structure and affinity of G-quadruplex DNA aptamers.
Journal of Nucleic Acids | 2012
Yuuya Kasahara; Masayasu Kuwahara
Specific binders comprised of nucleic acids, that is, RNA/DNA aptamers, are attractive functional biopolymers owing to their potential broad application in medicine, food hygiene, environmental analysis, and biological research. Despite the large number of reports on selection of natural DNA/RNA aptamers, there are not many examples of direct screening of chemically modified nucleic acid aptamers. This is because of (i) the inferior efficiency and accuracy of polymerase reactions involving transcription/reverse-transcription of modified nucleotides compared with those of natural nucleotides, (ii) technical difficulties and additional time and effort required when using modified nucleic acid libraries, and (iii) ambiguous efficacies of chemical modifications in binding properties until recently; in contrast, the effects of chemical modifications on biostability are well studied using various nucleotide analogs. Although reports on the direct screening of a modified nucleic acid library remain in the minority, chemical modifications would be essential when further functional expansion of nucleic acid aptamers, in particular for medical and biological uses, is considered. This paper focuses on enzymatic production of chemically modified nucleic acids and their application to random screenings. In addition, recent advances and possible future research are also described.
Bioorganic & Medicinal Chemistry Letters | 2016
Hidekazu Hoshino; Yuuya Kasahara; Hiroto Fujita; Masayasu Kuwahara; Kunihiko Morihiro; Shin-ichi Tsunoda; Satoshi Obika
Recently, 7-substituted 7-deazapurine nucleoside triphosphates and 5-substituted pyrimidine nucleoside triphosphates (dN(am)TPs) were synthesized to extend enzymatically using commercially available polymerase. However, extension was limited when we attempted to incorporate the substrates consecutively. To address this, we have produced a mutant polymerase that can efficiently accept the modified nucleotide with amphiphilic groups as substrates. Here we show that the KOD polymerase mutant, KOD exo(-)/A485L, had the ability to incorporate dN(am)TP continuously over 50nt, indicating that the mutant is sufficient for generating functional nucleic acid molecules.
Australian Journal of Chemistry | 2016
Kenta Hagiwara; Yuuya Kasahara; Hiroto Fujita; Masayasu Kuwahara
Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) is a kinetic capillary electrophoresis method used for the affinity analysis of DNA binding to proteins or ligands as well as a rapid selection of DNA aptamers. However, long DNA strands (100-mer or more) are generally difficult to analyse by this method owing to their poor peak separation. Herein, we report optimized conditions (use of a neutral phosphate buffer with an ionic strength of 0.074 as a binding buffer and use of an 80-cm fused silica capillary with a 75-μm internal diameter) for the peak separation of a 100-mer thrombin-binding DNA aptamer-target complex and its consequent enrichment using the NECEEM-based capillary electrophoresis–systematic evolution of ligands by exponential enrichment (CE-SELEX) method.
Bioorganic & Medicinal Chemistry Letters | 2013
Yuuya Kasahara; Yuuta Irisawa; Hiroaki Ozaki; Satoshi Obika; Masayasu Kuwahara
Analytical Chemistry | 2013
Yuuya Kasahara; Yuuta Irisawa; Hiroto Fujita; Aiko Yahara; Hiroaki Ozaki; Satoshi Obika; Masayasu Kuwahara
Bioorganic & Medicinal Chemistry Letters | 2010
Yuuya Kasahara; Shunsuke Kitadume; Kunihiko Morihiro; Masayasu Kuwahara; Hiroaki Ozaki; Hiroaki Sawai; Takeshi Imanishi; Satoshi Obika