論文 - 坂本　隆

Transcriptome analysis device based on liquid phase detection by fluorescently labeled nucleic acid probes

Yokokawa Ryuji, Tamaoki Soichiro, Sakamoto Takashi, Murakami Akira, Sugiyama Susumu

Biomedical microdevices ( Springer )  9 ( 6 ) 869 - 875   2007年  [査読有り]

DOI

Real-time monitoring of antibody secretion from B-cells on a microchip stimulated with a minute amount of mitogen

Munaka Tatsuya, Abe Hirohisa, Kanai Masaki, Sakamoto Takashi, Nakanishi Hiroaki, Shoji Shuichi, Kimura Shinya, Maekawa Taira, Murakami Akira

Analyst ( Royal Society of Chemistry )  132 ( 6 ) 512 - 514   2007年  [査読有り]

DOI

Homogeneous Fluorescence Assays for RNA Diagnosis by Pyrene-Conjugated 2′-O-Methyloligoribonucleotides

Sakamoto Takashi, Kobori Akio, Shigezawa Masako, Amitani Yasutaka, Higuchi Maiko, Murakami Akira (担当区分： 筆頭著者 )

Nucleosides, Nucleotides, and Nucleic Acids ( Taylor \& Francis )  26 ( 10-12 ) 1659 - 1664   2007年  [査読有り]

DOI

Highly-sensitive detection of immunoreactions by time-resolved fluorescence anisotropy analysis

Takashi Sakamoto, Nobuyuki Fujihara, Akio Kobori, Akira Murakami, Miharu Kishi, Koichi Yamagata

Polymer Preprints, Japan   55 ( 2 ) 4933   2006年

　概要を見る

Long-lifetime fluorophore is useful for the highly-sensitive detection of immunoreactions based on time-resolved fluorescence anisotropy analysis. We focused on pyrene-β-cyclodextrin conjugate (Py-β-CD) as a long-lifetime fluorescent label. Py-β-CD-NHS-ester was used to label human serum albumin (HSA). The steady-state fluorescence anisotropy of Py-β-CD-labeled HSA (Py-β-CD-HSA) was measured in the presence of various proteins. The fluorescence anisotropy of Py-β-CD-HSA increased upon the addition of anti-HSA monoclonal IgG or anti-HSA polyclonal IgG. On the other hand, the addition of anti-IgG IgG or HSA did not affect the fluorescence anisotropy of Py-β-CD-HSA. Time-resolved fluorescence anisotropy analysis of the Py-β-CD-HSA was carried out in the presence or absence of anti-HSA polyclonal IgG. The fluorescence anisotropy of the Py-β-CD-HSA from 0 to 200 nsec after pulse excitation significantly increased by the addition of anti-HSA polyclonal IgG. These results indicated that Py-β-CD-HSA bound to its specific IgG and that Py-β-CD-HSA was useful for highly-sensitive detection of immunoreactions based on time-resolved fluorescence anisotropy analysis.

Properties of novel antisense oligonucleotides containing 2′-O-modified adenosine with a photo-reactive group

Higuchi Maiko, Sakamoto Takashi, Kobori Akio, Murakami Akira

Nucleic Acids Symposium Series   50 ( 1 ) 301 - 2   2006年  [査読有り]

　概要を見る

In order to enhance the efficiency of antisense molecules for target RNA regulation, a novel photo-reactive antisense oligonucleotide was developed. We designed and synthesized a photo-reactive antisense oligonucleotide containing an adenosine in which 2'-OH was modified with 4,5',8-trimethylpsoralen (Ps) via an ethoxymethylene linkage (2'-Ps-eom). We evaluated the photo-cross-linking efficiency and sequence specificity toward complementary RNA (match-RNA). 2'-Ps-eom was selectively photo-cross-linked to the match-RNA. The photo-cross-linking efficiency was about 75% upon UVA-irradiation (365 nm) for 10 min. Previously, we reported oligonucleotides that had an adenosine anchoring Ps at 2'-O-position via a methylene linkage (2'-Ps-met). The photo-cross-linking efficiency of 2'-Ps-met and match-RNA was about 35% upon UVA-irradiation for 120 min. The photo-cross-linking efficiency of 2'-Ps-eom was dramatically enhanced in comparison with the one of 2'-Ps-met.

Solid-phase detection of RNA using bispyrene-modified RNA probe

Sakamoto Takashi, Kobori Akio, Murakami Akira (担当区分： 筆頭著者 )

Nucleic Acids Symposium Series   50 ( 1 ) 215 - 6   2006年  [査読有り]

　概要を見る

Bispyrene modified 2'-O-methyl oligoRNA (OMUpy2), which is a useful fluorescent probe for specific RNA detection, was immobilized on a glass substrate via various linker molecules to develop a more convenient RNA detection chip for gene expression study. As the fluorescence intensity of OMUpy2 enhanced greatly when it hybridized with its complementary RNA, it was expected that RNA detection by OMUpy2-immobilized chip (RNA-chip) required neither fluorescent labels to target nucleic acid nor washing protocol after the hybridization. As the linker molecules, oligo(dT), agarose, and poly(ethylene glycol) (PEG) were chosen, and the fluorescence from the chip was measured by a fluorescence microscope. Results indicated that the RNA-chip was able to detect the complementary oligoribonucleotide (cORN) without target labeling and washing protocols. Furthermore, the background emission was reduced as the length of the linker increased. In the case of PEG linker, 25-fold enhancement of the fluorescence intensity of the OMUpy2 was observed upon the addition of cORN.

Monitoring cellular events in living mast cells stimulated with an extremely small amount of fluid on a microchip

Munaka Tatsuya, Abe Hirohisa, Kanai Masaki, Sakamoto Takashi, Nakanishi Hiroaki, Yamaoka Tetsuji, Shoji Shuichi, Murakami Akira

Japanese journal of applied physics ( IOP Publishing )  45 ( 7L ) L634   2006年  [査読有り]

DOI

Real-time monitoring of antibody secretion from hybridomas on a microchip by time-resolved luminescence anisotropy analysis

Munaka Tatsuya, Abe Hirohisa, Kanai Masaki, Sakamoto Takashi, Nakanishi Hiroaki, Yamaoka Tetsuji, Shoji Shuichi, Murakami Akira

Analytical biochemistry ( Elsevier )  353 ( 1 ) 1 - 6   2006年  [査読有り]

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Development of the cell analysis chip (II)

Tatsuya Munaka, Masaki Kanai, Hirohisa Abe, Hiroaki Nakanishi, Takashi Sakamoto, Akio Kobori, Tetsuji Yamaoka, Shuichi Shoji, Akira Murakami

Polymer Preprints, Japan   54 ( 1 ) 2329   2005年  [査読有り]

　概要を見る

In recent years, cell analysis has attracted increased attention in bioanalytical and biotechnological applications. On the other hand, microchip techniques, which are called micro total analysis systems (μTAS), have been developed especially in chemical and biochemical analysis fields. We took advantage of a feature of μ TAS and developed a system that can detect a small amount of the protein released by a cell without bound/free (B/F) separation protocol and with high sensitivity. In this report, we describe the continuous monitoring of immunoglobulin G (lgG) production of hybridoma cells stimulated by the serum.

Analytical method for estimation of kinetics of oligonucleotide/RNA hybridization using fluorescence depolarization spectroscopy

Sakamoto Takashi, Mahara Atsushi, Iwase Reiko, Yamaoka Tetsuji, Murakami Akira (担当区分： 筆頭著者 )

Analytical biochemistry ( Academic Press )  340 ( 2 ) 369 - 372   2005年  [査読有り]

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Transcriptome Analyzing Chip Based on Liquid-phase Detection Using Fluorescence Labeled Nucleic Acid Probe

Tamaoki S, Sugiyama S, Sakamoto T, Murakami A

PROCEEDINGS OF THE SENSOR SYMPOSIUM ON SENSORS MICROMACHINES AND APPLIED SYSTEMS   22   270   2005年  [査読有り]

Evaluation of dynamic features of Escherichia coli 16S ribosomal RNA in homogeneous physiological solution

Sakamoto Takashi, Mahara Atsushi, Yamagata Koichi, Iwase Reiko, Yamaoka Tetsuji, Murakami Akira (担当区分： 筆頭著者 )

Biophysical journal ( Elsevier )  89 ( 6 ) 4122 - 4128   2005年  [査読有り]

DOI

Luminescence anisotropy-based detection of nucleic acids and proteins using long-lifetime Ru(II) complex as a luminescent label.

Takashi Sakamoto, Atsushi Mahara, Kobori Akio, Tetsuji Yamaoka, Akira Murakami (担当区分： 筆頭著者 )

Nucleic acids symposium series (2004)   49 ( 49 ) 203 - 204   2005年  [査読有り]

　概要を見る

Luminescence anisotropy-based methods are powerful tool for the detection of biomolecules in homogeneous physiological media without Bound/Free separation. However, analyses based on the method are always disturbed by the autofluorescence from biological specimen. This disturbance limits the sensitivity of the method. To improve the detection limit of the method, a long-lifetime luminophore was adopted as the label. Because of the lifetime of the autofluorescence is below 10 ns, it is expected that the use of the long-lifetime luminophore enables us to avoid the disturbance by adopting time-resolved luminescence anisotropy measurement. To verify this concept, oligodeoxyribonucleotides and Staphylococcus aureus protein A were labeled with Ru(II) complex (tau approximately 500 nsec) and used for the detection of Escherichia coli 16S ribosomal RNA (16S rRNA) and immunoglobulin G (IgG), respectively. Results indicated that the methods were useful for the detection of rRNA and IgG without disturbance of the autofluorescence.

Time-resolved luminescence anisotropy-based detection of immunoglobulin G using long-lifetime Ru (II) complex-labeled protein A

Sakamoto Takashi, Mahara Atsushi, Munaka Tatsuya, Yamagata Koich, Iwase Reiko, Yamaoka Tetsuji, Murakami Akira (担当区分： 筆頭著者 )

Analytical biochemistry ( Academic Press )  329 ( 1 ) 142 - 144   2004年  [査読有り]

DOI

Transcriptome analysis using fluorescence-labeled oligonucleotide.

Naohiko Shimada, Atsushi Mahara, Takashi Sakamoto, Tetsuji Yamaoka, Akira Murakami

Nucleic acids symposium series (2004)   48 ( 48 ) 301 - 302   2004年  [査読有り]

　概要を見る

In our previous studies, the fluorescence intensity at 480 nm of the bispyrene-labeled 2'-O-methyl-oligoribonucleotide (OMUpy2) was drastically enhanced only when it was hybridized with its complementary RNA. In this study, we demonstrated the potential of the OMUpy2 for transcriptome analysis in the cell free system and in the living cell. For the analysis in the cell free system, the micro-chamber was designed and used to evaluate the detection limit of the target RNA. The detection limit of oligo-RNA was approximately 15 fmol. For the analysis of the living cells, C-OMUpy2 was introduced to the cervical carcinoma cells, C4II, and the fluorescence from C-OMUpy2 was monitored. The fluorescence from the cells began to be observed 5 min after the serum stimulation, and the fluorescence was decreased after 20 min. These results seemed to be consistent with the results from the RT-PCR analysis and suggested that OMUpy2 could be a useful tool for the transcriptome analysis.

In situ Cell Monitoring on a Microchip using Time-Resolved Fluorescence Anisotropy Analysis

Sakamoto Takashi, Mahara Atsushi, Yamayoshi Asako

生物物理 ( 一般社団法人 日本生物物理学会 )  43   S222   2003年10月  [査読有り]

DOI

RNAの機能・構造解析に用いる螢光RNAプローブの開発

村上 章, 坂本 隆, 馬原 淳

化学と生物 ( Japan Society for Bioscience, Biotechnology, and Agrochemistry )  41 ( 8 ) 549 - 556   2003年08月

DOI

Detection of acceptor sites for antisense oligonucleotides on native folded RNA by fluorescence-labeled oligonucleotide.

Atsushi Mahara, Takashi Sakamoto, Tatsuya Munaka, Reiko Iwase, Tetsuji Yamaoka, Akira Murakami

Nucleic acids research. Supplement (2001)   3 ( 3 ) 73 - 74   2003年  [査読有り]

　概要を見る

Pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy) and 5'-Ru(II) complex labeled oligodeoxyribonucleotie (Ru-probe) were prepared as the fluorescence probes to detect acceptor sites of antisense molecules on native folded RNA under the physiological condition. OMUpy showed the remarkable increase of the fluorescence intensity (334-fold at 375 nm) only when hybridized with complementary oligoRNA. When OMUpy was applied to E. coli 16S-rRNA, the fluorescence intensities were increased in a sequence specific manner. The rotational correlation time of Ru-probe complementary to 16S-rRNA were largely dependent on the sequence of Ru-probe. These results suggest that the accessibility of the antisense molecules for RNA can be evaluated by those fluorescent probes.

Detection of acceptor sites for antisense oligonucleotides on native folded RNA by fluorescence spectroscopy

Mahara Atsushi, Iwase Reiko, Sakamoto Takashi, Yamaoka Tetsuji, Yamana Kazushige, Murakami Akira

Bioorganic & medicinal chemistry ( Elsevier )  11 ( 13 ) 2783 - 2790   2003年  [査読有り]

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Bispyrene-conjugated 2'-O-methyloligonucleotide as a highly specific RNA-recognition probe.

Atsushi Mahara, Reiko Iwase, Takashi Sakamoto, Kazushige Yamana, Tetsuji Yamaoka, Akira Murakami

Angewandte Chemie (International ed. in English)   41 ( 19 ) 3648 - 50   2002年10月