東北大学研究者紹介

顔写真

サトウ　ユウスケ

佐藤　雄介

Yusuke Sato

所属

大学院理学研究科　化学専攻　無機・分析化学講座（分析化学研究室）

職名

准教授

学位

博士（理学）（東北大学）
修士（理学）（東北大学）

researchmap

https://researchmap.jp/read0154897

J-GLOBAL ID

201101022046839498

委員歴 2

日本分析化学会東北支部　日本分析化学会東北支部幹事

2010年4月～継続中
日本分析化学会東北支部　日本分析化学会東北支部若手代表

2016年4月～ 2022年3月

所属学協会 2

日本化学会
日本分析化学会

研究キーワード 2

生体機能関連化学
分析化学

研究分野 1

ナノテク・材料 / 分析化学 /

受賞 5

科学技術分野の文部科学大臣表彰若手科学者賞

2021年4月
平成29年度進歩賞

2018年3月　日本化学会　核酸構造特異的な蛍光プローブの創製と分析化学的応用
日本分析化学会2014年度奨励賞

2014年9月18日　日本分析化学会
第２８回井上研究奨励賞

2012年2月3日　井上科学振興財団
2009年度日本分析化学会東北支部奨励賞

2009年12月　日本分析化学会東北支部

論文 62

Evaluation of respiratory and secretory activities of multicellular spheroids via electrochemiluminescence imaging

Kaoru Hiramoto, Keika Komatsu, Ryota Shikuwa, An Konno, Yusuke Sato, Ayumi Hirano-Iwata, Kosuke Ino, Hitoshi Shiku

Electrochimica Acta　458　142507-142507　2023年8月
出版者・発行元：Elsevier BV
DOI： 10.1016/j.electacta.2023.142507 　

ISSN：0013-4686
Synthetic DNA binders for fluorescence sensing of thymine glycol-containing DNA duplexes and inhibition of endonuclease activity 査読有り

Yusuke Sato, Yoshihide Takaku, Toshiaki Nakano, Ken Akamatsu, Dai Inamura, Seiichi Nishizawa

Chemical communication　in press　2023年5月

DOI： 10.1039/D3CC01501G 　
Cationic Oligopeptides with Amino Groups as Synthetic Nucleolar Localization Signals for the Rational Design of Nucleolus-Staining Probes

Michiyuki Suzuki, Yusuke Sato, Nao Togashi, Seiichi Nishizawa

ACS Omega　8　(10)　9592-9596　2023年2月28日
出版者・発行元：American Chemical Society (ACS)
DOI： 10.1021/acsomega.3c00116 　

ISSN：2470-1343

eISSN：2470-1343
Inhibition of SARS-CoV-2 nucleocapsid protein–RNA interaction by guanosine oligomeric RNA

Ryoya Sekine, Satsuki Tsuno, Hayato Irokawa, Kazuhiro Sumitomo, Tianxue Han, Yusuke Sato, Seiichi Nishizawa, Kouki Takeda, Shusuke Kuge

The Journal of Biochemistry　173　(6)　447-457　2023年2月7日
出版者・発行元：Oxford University Press (OUP)
DOI： 10.1093/jb/mvad008 　

ISSN：0021-924X

eISSN：1756-2651

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Abstract The interaction of the β-coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein with genomic RNA is initiated by specific RNA regions and subsequently induces the formation of a continuous polymer with characteristic structural units for viral formation. We hypothesized that oligomeric RNAs, whose sequences are absent in the 29.9-kb genome sequence of SARS-CoV-2, might affect RNA–N protein interactions. We identified two such hexameric RNAs, In-1 (CCGGCG) and G6 (GGGGGG), and investigated their effects on the small filamentous/droplet-like structures (&lt; a few μm) of N protein–genomic RNA formed by liquid–liquid phase separation. The small N protein structures were sequence-specifically enhanced by In-1, whereas G6 caused them to coalesce into large droplets. Moreover, we found that a guanosine 12-mer (G12, GGGGGGGGGGGG) expelled preexisting genomic RNA from the small N protein structures. The presence of G12 with the genomic RNA suppressed the formation of the small N protein structures, and alternatively apparently altered phase separation to induce the formation of large droplets with unclear phase boundaries. We showed that the N-terminal RNA-binding domain is required for the stability of the small N protein structures. Our results suggest that G12 may be a strong inhibitor of the RNA–N protein interaction.
Self-Assembly and Disassembly of Membrane Curvature-Sensing Peptide-Based Deep-Red Fluorescent Probe for Highly Sensitive Sensing of Exosomes

Kaito Ohira, Yusuke Sato, Seiichi Nishizawa

ACS Sensors　8　(2)　522-526　2023年1月25日
出版者・発行元：American Chemical Society (ACS)
DOI： 10.1021/acssensors.2c02498 　

ISSN：2379-3694

eISSN：2379-3694
Classical thiazole orange and its regioisomer as fluorogenic probes for nucleolar RNA imaging in living cells

Mengmeng He, Yusuke Sato, Seiichi Nishizawa

The Analyst　148　(3)　636-642　2023年
出版者・発行元：Royal Society of Chemistry (RSC)
DOI： 10.1039/d2an01804g 　

ISSN：0003-2654

eISSN：1364-5528

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Thiazole orange (TO) performs as a promising fluorogenic dye for nucleolar RNA imaging in living cells. More interestingly, 2TO, a regioisomer of TO, performs much better and has superior selectivity for RNA in both solution and living cells.
Kinetic analysis of highly effective triplex formation between a small molecule–peptide nucleic acid conjugate probe and the influenza A virus RNA promoter region at neutral pH

Chioma Uche Okeke, Hiromasa Miura, Yusuke Sato, Seiichi Nishizawa

Organic & Biomolecular Chemistry　21　(16)　3402-3410　2023年
出版者・発行元：Royal Society of Chemistry (RSC)
DOI： 10.1039/d3ob00262d 　

ISSN：1477-0520

eISSN：1477-0539

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The binding of PNA–small molecule conjugate probes to IAV RNA was examined by means of a stopped-flow technique. It is highly likely that conjugation is the key factor to realize the effective triplex formation of PNA with natural nucleobases at neutral pH.
Multiplex microRNA detection

Yusuke Sato

Analytical Sciences　38　(9)　1123-1124　2022年8月29日
出版者・発行元：Springer Science and Business Media LLC
DOI： 10.1007/s44211-022-00152-0 　

ISSN：0910-6340

eISSN：1348-2246
Bright and Light-Up Sensing of Benzo[c,d]indole-oxazolopyridine Cyanine Dye for RNA and Its Application to Highly Sensitive Imaging of Nucleolar RNA in Living Cells

Kei Higuchi, Yusuke Sato, Nao Togashi, Michiyuki Suzuki, Yukina Yoshino, Seiichi Nishizawa

ACS Omega　7　(27)　23744-23748　2022年6月29日
出版者・発行元：American Chemical Society (ACS)
DOI： 10.1021/acsomega.2c02408 　

ISSN：2470-1343

eISSN：2470-1343
Fluorescence Sensing of the Panhandle Structure of the Influenza A Virus RNA Promoter by Thiazole Orange Base Surrogate-Carrying Peptide Nucleic Acid Conjugated with Small Molecule. 国際誌

Yusuke Sato, Hiromasa Miura, Takaaki Tanabe, Chioma Uche Okeke, Akiko Kikuchi, Seiichi Nishizawa

Analytical chemistry　94　(22)　7814-7822　2022年6月7日

DOI： 10.1021/acs.analchem.1c05488 　

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We have developed a new class of triplex-forming peptide nucleic acid (PNA)-based fluorogenic probes for sensing of the panhandle structure of the influenza A virus (IAV) RNA promoter region. Here, a small molecule (DPQ) capable of selectively binding to the internal loop structure was conjugated with triplex-forming forced intercalation of the thiazole orange (tFIT) probe with natural PNA nucleobases. The resulting conjugate, tFIT-DPQ, showed a significant light-up response (83-fold) upon strong (Kd = 107 nM) and structure-selective binding to the IAV RNA promoter region under physiological conditions (pH 7.0, 100 mM NaCl). We demonstrated the conjugation of these two units through the suitable spacer was key to show useful binding and fluorogenic signaling functions. tFIT-DPQ facilitated the sensitive and selective detection of IAV RNA based on its binding to the promoter region. Furthermore, we found that tFIT-DPQ could work as a sensitive indicator for screening of test compounds targeting the IAV RNA promoter region in the fluorescence indicator displacement assay.
Triplex-Forming Peptide Nucleic Acid Probes Having Cyanine Base Surrogates for Fluorogenic Sensing of Double-Stranded RNA

Seiichi NISHIZAWA, Takaya SATO, En Ting Tabitha LEE, Naonari SAKAMOTO, Toshiki CHIBA, Takaaki TANABE, Yukina YOSHINO, Yuki TAKAHASHI, Yusuke SATO

BUNSEKI KAGAKU　71　(3)　133-144　2022年3月5日
出版者・発行元：Japan Society for Analytical Chemistry
DOI： 10.2116/bunsekikagaku.71.133 　

ISSN：0525-1931
Molecular Design of Fluorogenic Probes for Targeting rRNA: Indicator in FID Assay and Dye for Imaging of Nucleolar RNA in Living Cells

Seiichi NISHIZAWA, En Ting Tabitha LEE, Yukina YOSHINO, Sayaka YAJIMA, Masafumi ROKUGAWA, Yusuke SATO

BUNSEKI KAGAKU　70　(12)　703-714　2021年12月5日
出版者・発行元：Japan Society for Analytical Chemistry
DOI： 10.2116/bunsekikagaku.70.703 　

ISSN：0525-1931
Spectroscopic, thermodynamic and kinetic analysis of selective triplex formation by peptide nucleic acid with double‐stranded <scp>RNA</scp> over its <scp>DNA</scp> counterpart

Takaya Sato, Yusuke Sato, Seiichi Nishizawa

Biopolymers　113　(1)　2021年9月3日
出版者・発行元：Wiley
DOI： 10.1002/bip.23474 　

ISSN：0006-3525

eISSN：1097-0282

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Abstract Unlike conventional triplex‐forming oligonucleotide (TFO), triplex‐forming peptide nucleic acid (PNA) can tightly bind with double‐stranded RNA (dsRNA) than double‐stranded DNA (dsDNA). Here, we performed spectroscopic, thermodynamic and kinetic experiments for triplex formation by PNA to examine different binding behaviors between PNA − dsRNA and PNA − dsDNA triplexes. We found 9‐mer PNA (cytosine content of 66%) formed the thermally stable triplex with dsRNA compared to dsDNA over a wide range of pH (5.5‐8.0), salt concentration (50‐500 mM NaCl). Both the calorimetric binding constant and the association rate constant for dsRNA were larger than those for dsDNA, indicating the favorable association process for the PNA − dsRNA triplex formation. Comparison with the DNA/RNA heteroduplexes revealed that the DNA strand was detrimental to the triplex stability for PNA, a contrasting result for conventional TFO. The keys underlying the difference in the triplex formation of PNA with different duplexes appear to be the conformational adoptability and the geometric compatibility of PNA to fit the deep, narrow major groove of dsRNA and the helical rigidity difference of the duplexes. Our results emphasize the importance of both the sugar puckering of the duplex and the appropriate conformational flexibility of PNA for the triplex formation.
Fluorogenic Indicator for Targeting HIV-1 TAR RNA: Evaluation for Binging and Signaling Functions of Monomethine Cyanine Dye Thiazole Orange and Its Application to FID Assay

Yoshiko ITO, Yusuke SATO, Norio TERAMAE, Seiichi NISHIZAWA

BUNSEKI KAGAKU　70　(3)　149-157　2021年3月5日
出版者・発行元：Japan Society for Analytical Chemistry
DOI： 10.2116/bunsekikagaku.70.149 　

ISSN：0525-1931
Deep-red fluorogenic cyanine dyes carrying an amino group-terminated side chain for improved RNA detection and nucleolar RNA imaging

Yusuke Sato, Yugo Igarashi, Michiyuki Suzuki, Kei Higuchi, Seiichi Nishizawa

RSC Advances　11　(56)　35436-35439　2021年
出版者・発行元：Royal Society of Chemistry (RSC)
DOI： 10.1039/d1ra05872j 　

eISSN：2046-2069

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The introduction of an amino-group-terminated side chain into deep-red emissive benzo[c,d]indole–quinoline monomethine cyanine dye has led to the improved detection of RNAs as well as the imaging of nucleolar RNAs in cells.
Chloro-Substituted Naphthyridine Derivative and Its Conjugate with Thiazole Orange for Highly Selective Fluorescence Sensing of an Orphan Cytosine in the AP Site-Containing Duplexes

Chun-xia Wang, Yusuke Sato, Takashi Sugimoto, Norio Teramae, Seiichi Nishizawa

Applied Sciences　10　(12)　4133-4133　2020年6月16日
出版者・発行元：MDPI AG
DOI： 10.3390/app10124133 　

eISSN：2076-3417

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Fluorescent probes with the binding selectivity to specific structures in DNAs or RNAs have gained much attention as useful tools for the study of nucleic acid functions. Here, chloro-substituted 2-amino-5,7-dimethyl-1,8-naphthyridine (ClNaph) was developed as a strong and highly selective binder for target orphan cytosine opposite an abasic (AP) site in the DNA duplexes. ClNaph was then conjugated with thiazole orange (TO) via an alkyl spacer (ClNaph–TO) to design a light-up probe for the detection of cytosine-related mutations in target DNA. In addition, we found the useful binding and fluorescence signaling of the ClNaph–TO conjugate to target C in AP site-containing DNA/RNA hybrid duplexes with a view toward sequence analysis of microRNAs.
Design of Fluorescent Peptide Nucleic Acid Probes Carrying Cyanine Dyes for Targeting Double-Stranded RNAs for Analytical Applications

Yusuke Sato

Bulletin of the Chemical Society of Japan　93　(3)　406-413　2020年3月15日
出版者・発行元：The Chemical Society of Japan
DOI： 10.1246/bcsj.20190361 　

ISSN：0009-2673

eISSN：1348-0634
Conjugating pyrene onto PNA-based fluorescent probes for improved detection selectivity toward double-stranded siRNA

Yusuke Sato, Yuki Takahashi, Takaaki Tanabe, Seiichi Nishizawa

Organic & Biomolecular Chemistry　18　(21)　4009-4013　2020年
出版者・発行元：Royal Society of Chemistry (RSC)
DOI： 10.1039/d0ob00794c 　

ISSN：1477-0520

eISSN：1477-0539

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PNA-based fluorescent probes conjugated with the pyrene unit exhibited the improved detection selectivity for double-stranded siRNA.
Amphipathic helical peptide-based fluorogenic probes for a marker-free analysis of exosomes based on membrane-curvature sensing

Yusuke Sato, Kazuki Kuwahara, Kenta Mogami, Kenta Takahashi, Seiichi Nishizawa

RSC Advances　10　(63)　38323-38327　2020年
出版者・発行元：Royal Society of Chemistry (RSC)
DOI： 10.1039/d0ra07763a 　

eISSN：2046-2069

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Fluorogenic probes based on membrane curvature sensing-amphipathic helical peptides have been developed for a marker-free exosome analysis.
Small molecule–PNA oligomer conjugates for rRNA A-site at neutral pH for FID assays

En Ting Tabitha Lee, Yusuke Sato, Seiichi Nishizawa

Chemical Communications　56　(95)　14976-14979　2020年
出版者・発行元：Royal Society of Chemistry (RSC)
DOI： 10.1039/d0cc06084d 　

ISSN：1359-7345

eISSN：1364-548X

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A conjugation strategy was used to synthesize light-up probes for the bacterial rRNA A-site with strong binding affinity and selectivity.
Deep-Red Light-up Signaling of Benzo[c,d]indole–Quinoline Monomethine Cyanine for Imaging of Nucleolar RNA in Living Cells and for Sequence-Selective RNA Analysis

Yukina Yoshino, Yusuke Sato, Seiichi Nishizawa

Analytical Chemistry　91　(22)　14254-14260　2019年10月9日
出版者・発行元：American Chemical Society (ACS)
DOI： 10.1021/acs.analchem.9b01997 　

ISSN：0003-2700

eISSN：1520-6882
Strong binding and off-on signaling functions of deep-red fluorescent TO-PRO-3 for influenza A virus RNA promoter region 査読有り

Y. Sato, Y. Aiba, S. Yajima, T. Tanabe, K. Higuchi, S. Nishizawa

ChemBioChem　20　2752-2756　2019年10月
Programmable RNA Detection with a Fluorescent RNA Aptamer Using Optimized Three-way Junction Formation 査読有り

Y. Furuhata, M. Kobayashi, R. Maruyama, Y. Sato, K. Makino, T. Michiue, H. Yui, S. Nishizawa, K. Yoshimoto

RNA　25　(5)　590-599　2019年2月
出版者・発行元：None
DOI： 10.1261/rna.069062.118 　

ISSN：1355-8382

eISSN：1469-9001
Enhanced binding affinity of siRNA overhang-binding fluorescent probes by conjugation with cationic oligopeptides for the improved analysis of siRNA delivery process 査読有り

Y. Sato, M. Kaneko, T. Sato, S. Nakata, Y. Takahashi, S. Nishizawa

ChemBioChem　20　408-414　2019年2月
Trimethine cyanine dyes as deep-red fluorescent indicators with high selectivity to the internal loop of the bacterial A-site RNA 査読有り

Y. Sato, S. Yajima, A. Taguchi, K. Baba, M. Nakagomi, Y. Aiba, S. Nishizawa

Chem. Commun.　55　3183-3186　2019年1月
Design of a fluorogenic PNA probe capable of simultaneous recognition of 3’-overhang and double-stranded sequences of small interfering RNAs 査読有り

T. Tanabe, T. Sato, Y. Sato, S. Nishizawa

RSC Advances　8　42095-42099　2018年12月
RNA結合性蛍光プローブの設計・合成と小分子RNA解析査読有り

佐藤雄介, 佐藤貴哉, 西澤精一

分析化学　67　531-540　2018年9月
Fluorescent trimethylated naphthyridine derivative with an aminoalkyl side chain as the tightest non-aminoglycoside ligand for the bacterial A-site RNA 査読有り

Y. Sato, M. Rokugawa, S. Ito, S. Yajima, H. Sugawara, N. Teramae, S. Nishizawa

Chem. Eur. J.　24　13862-13870　2018年9月
Effect of Cavity Size of Mesoporous Silica on Short DNA Duplex Stability 査読有り

Tsubasa Masuda, Yuuta Shibuya, Shota Arai, Sayaka Kobayashi, Sotaro Suzuki, Jun Kijima, Tetsuji Itoh, Yusuke Sato, Seiichi Nishizawa, Akira Yamaguchi

Langmuir　34　(19)　5545-5550　2018年5月15日
出版者・発行元：American Chemical Society
DOI： 10.1021/acs.langmuir.8b00437 　

ISSN：1520-5827 0743-7463

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We studied the stabilities of short (4- and 3-bp) DNA duplexes within silica mesopores modified with a positively charged trimethyl aminopropyl (TMAP) monolayer (BJH pore diameter 1.6-7.4 nm). The DNA fragments with fluorescent dye were introduced into the pores, and their fluorescence resonance energy transfer (FRET) response was measured to estimate the structuring energies of the short DNA duplexes under cryogenic conditions (temperature 233-323 K). The results confirmed the enthalpic stability gain of the duplex within size-matched pores (1.6 and 2.3 nm). The hybridization equilibrium constants found for the size-matched pores were 2 orders of magnitude larger than those for large pores (≥3.5 nm), and this size-matching effect for the enhanced duplex stability was explained by a tight electrostatic interaction between the duplex and the surface TMAP groups. These results indicate the requirement of the precise regulation of mesopore size to ensure the stabilization of hydrogen-bonded supramolecular assemblies.
Highly sensitive programmable RNA detection using fluorescent RNA aptamer 査読有り

Furuhata Yuichi, Kobayahshi Mizuki, Maruyama Ryo, Sato Yusuke, Makino Kurumi, Michiue Tatsuo, Yui Hiroharu, Nishizawa Seiichi, Yoshimoto Keitaro

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY　255　2018年3月18日
出版者・発行元：None
ISSN：0065-7727
Improved boronate affinity electrophoresis by optimization of the running buffer for a single-step separation of piRNA from mouse testis total RNA 査読有り

Yusuke Sato, Daijiro Iwasawa, Kuo Ping Hui, Rena Nakagomi, Seiichi Nishizawa

Analytical Sciences　34　(5)　627-630　2018年
出版者・発行元：Japan Society for Analytical Chemistry
DOI： 10.2116/analsci.17N024 　

ISSN：1348-2246 0910-6340

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Here we examined optimization of the running buffer in boronate affinity electrophoresis for improved separation of PIWI-interacting RNA (piRNA) with 2'-O-methylated ribose in 3'-terminal nucleotide. The use of Good's buffer, such as HEPES, significantly increased the separation efficiency for piRNA over normal RNA with free 3'-terminal ribose, and retained an ability to resolve the difference by at least 4-nucleotide lengths in the target piRNAs. We also demonstrated a single-step separation of piRNA from mouse testis total RNA.
Red-emissive triplex-forming PNA probes carrying cyanine base surrogates for fluorescence sensing of double-stranded RNA 査読有り

Toshiki Chiba, Takaya Sato, Yusuke Sato, Seiichi Nishizawa

ORGANIC & BIOMOLECULAR CHEMISTRY　15　(37)　7765-7769　2017年10月
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/c7ob02077e 　

ISSN：1477-0520

eISSN：1477-0539

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Red-emissive fluorescent probes have been developed by integration of quinoline blue or thiazole red as the base surrogate into triplex-forming PNAs, allowing selective sensing of a sequence of double-stranded RNA.
Optimization of the Alkyl Linker of TO Base Surrogate in Triplex-Forming PNA for Enhanced Binding to Double-Stranded RNA 査読有り

Takaya Sato, Yusuke Sato, Seiichi Nishizawa

CHEMISTRY-A EUROPEAN JOURNAL　23　(17)　4079-4088　2017年3月
出版者・発行元：WILEY-V C H VERLAG GMBH
DOI： 10.1002/chem.201604676 　

ISSN：0947-6539

eISSN：1521-3765

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A series of triplex-forming peptide nucleic acid (TFP) probes carrying a thiazole orange (TO) base surrogate through an alkyl linker was synthesized, and the interactions between these so-called tFIT probes and purine-rich sequences within double-stranded RNA (dsRNA) were examined. We found that the TO base surrogate linker significantly affected both the binding affinity and the fluorescence response upon triplex formation with the target dsRNA. Among the probes examined, the TO base surrogate connected through the propyl linker in the tFIT probes increased the binding affinity by a factor of ten while maintaining its function as the fluorescent universal base. Isothermal titration calorimetry experiments revealed that the increased binding affinity resulted from the gain in the binding enthalpy, which could be explained by the enhanced pstacking interaction between the TO base surrogate and the dsRNA part of the triplex. We expect that these results will provide a molecular basis for designing strong binding tFIT probes for fluorescence sensing of various kinds of purinerich dsRNAs sequences including those carrying a pyrimidine-purine inversion. The obtained data also offers a new insight into further development of the universal bases incorporated in TFP.
Triplex-Forming Peptide Nucleic Acid Probe Having Thiazole Orange as a Base Surrogate for Fluorescence Sensing of Double-stranded RNA 査読有り

Takaya Sato, Yusuke Sato, Seiichi Nishizawa

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY　138　(30)　9397-9400　2016年8月
出版者・発行元：AMER CHEMICAL SOC
DOI： 10.1021/jacs.6b05554 　

ISSN：0002-7863

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We have developed a new fluorescent sensing probe for double-stranded RNA (dsRNA) by integrating thiazole orange (TO) as a base surrogate into triplex-forming PNA. Our probe forms the thermally stable triplex with the target dsRNA at acidic pH; and the triplex formation is accompanied by the remarkable light-up response of the TO unit. The binding of our probe to the target dsRNA proceeds very rapidly, allowing real-time monitoring of the triplex formation. Importantly, we found the TO base surrogate in our probe functions as a universal base for the base pair opposite the TO unit in the triplex formation. Furthermore, the TO unit is significantly more responsive for the fully matched dsRNA sequence compared to the mismatch-containing sequences, which enables the analysis of the target dsRNA sequence at the single-base pair resolution. The binding and sensing functions of our probe are described for the development of fluorescent probes applicable to sensing biologically relevant dsRNA.
Pteridine derivatives modified with a guanidine for binding and sensing an orphan guanine in RNA duplexes 査読有り

Yusuke Sato, Takahiro Asami, Yu Toriyabe, Takaya Sato, Norio Teramae, Seiichi Nishizawa

Chemistry Letters　45　(8)　982-984　2016年
出版者・発行元：Chemical Society of Japan
DOI： 10.1246/cl.160425 　

ISSN：1348-0715 0366-7022

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We report on a fluorescent pteridine derivative modified with a guanidino group for the selective detection of an orphan guanine in RNA duplexes with a dissociation constant of 31 ± 7 nM (pH 7.0, T = 20°C). The significant effect of the guanidino group attached to the pteridine ring on the binding selectivity and affinity is discussed based on the examination of thermodynamic parameters.
Lysine linkage in abasic site-binding ligand-thiazole orange conjugates for improved binding affinity to orphan nucleobases in DNA/RNA hybrids 査読有り

Yusuke Sato, Hiroki Saito, Daisuke Aoki, Norio Teramae, Seiichi Nishizawa

CHEMICAL COMMUNICATIONS　52　(100)　14446-14449　2016年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/c6cc07236d 　

ISSN：1359-7345

eISSN：1364-548X

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Introduction of lysine linkage in the conjugate between abasic site-binding ligands and thiazole orange significantly improved the binding affinity for target orphan adenine or uracil nucleobase in DNA/RNA hybrids.
Fluorescence Imaging of siRNA Delivery by Peptide Nucleic Acid-based Probe 査読有り

Takaya Sato, Yusuke Sato, Kenta Iwai, Shusuke Kuge, Norio Teramae, Seiichi Nishizawa

ANALYTICAL SCIENCES　31　(4)　315-320　2015年4月
出版者・発行元：JAPAN SOC ANALYTICAL CHEMISTRY
DOI： 10.2116/analsci.31.315 　

ISSN：0910-6340

eISSN：1348-2246

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We report on the use of a peptide nucleic acid (PNA)-based fluorescent probe for the analysis of siRNA delivery to living cells. The probe, Py-AA-TO, possesses thiazole orange (TO) and pyrene moieties in the C- and N-termini of PNA, and can function as a light-up probe capable of selective binding to 3'-overhanging nucleotides of target siRNAs. The affinity-labeling of the siRNAs with Py-AA-TO facilitates fluorescence imaging of cellular uptake of polymer-based carriers encapsulating the siRNAs (polyplexes) through endocytosis and subsequent sequestration into lysosome. In addition, flow cytometric measurements reveal that the monitoring of Py-AA-TO fluorescence inside the cells is successfully applicable to the analysis of the polyplex disassembly. These promising functions of Py-AA-TO are presented and discussed as a basis for the design of molecular probes for fluorescent imaging and quantitative analysis of the siRNA delivery process.
A label-free and sensitive fluorescent method for the detection of uracil-DNA glycosylase activity 査読有り

Jing Tao, Panshu Song, Yusuke Sato, Seiichi Nishizawa, Norio Teramae, Aijun Tong, Yu Xiang

CHEMICAL COMMUNICATIONS　51　(5)　929-932　2015年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/c4cc06170e 　

ISSN：1359-7345

eISSN：1364-548X

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The activity of uracil-DNA glycosylase (UDG), an enzyme in the base excision repair, is detected at a high sensitivity by a DNA substrate containing only one uracil through a label-free fluorescent approach, which is also successfully applied for the measurement of UDG inhibitors.
Synthetic fluorescent probes capable of selective recognition of 3 '-overhanging nucleotides for siRNA delivery imaging 査読有り

Takaya Sato, Yusuke Sato, Kenta Iwai, Shusuke Kuge, Seiichi Nishizawa, Norio Teramae

CHEMICAL COMMUNICATIONS　51　(8)　1421-1424　2015年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/c4cc08800j 　

ISSN：1359-7345

eISSN：1364-548X

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Peptide nucleic acid (PNA)-thiazole orange (TO) conjugates are developed as fluorescent probes capable of selective recognition of 3'-overhanging nucleotides of siRNAs for an accurate analysis of the siRNA delivery process.
Trinucleotide duplex formation inside a confined nanospace under supercooled conditions 査読有り

Hiroyuki Arafune, Akira Yamaguchi, Manato Namekawa, Yusuke Sato, Tetsuji Itoh, Ryoko Yoshida, Norio Teramae

NATURE COMMUNICATIONS　5　2014年10月
出版者・発行元：NATURE PUBLISHING GROUP
DOI： 10.1038/ncomms6151 　

ISSN：2041-1723

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Self-assembly of nucleotides of fewer than three base pairs is often found in protein-nucleotide conjugations, despite their energetic instability, and is regarded as the potential starting point for the creation of artificial hydrogen-bonded supramolecular complexes. Here we report duplex formation of 3-mer DNA fragments confined within silica mesopores modified with a positively charged trimethyl aminopropyl monolayer, and their further stabilization under supercooled conditions (T<273 K). We load 3-mer DNA fragments with donor-or acceptor-dye into modified silica mesopores and examine their hybridization behaviours using FRET measurements. The FRET results clearly reveal that efficient duplex formation through at least two A-T base pairs can be achieved at 233 K. Enthalpy changes for duplex formation are found to be nearly equal between complementary and single-mismatched 3-mer DNA duplexes. These results confirm confined mesoscale cavities to be a novel low-temperature reaction space for hydrogen-bonded supramolecular complexes.
Abasic site-binding ligands conjugated with cyanine dyes for "off-on" fluorescence sensing of orphan nucleobases in DNA duplexes and DNA-RNA hybrids 査読有り

Yusuke Sato, Megumi Kudo, Yu Toriyabe, Shota Kuchitsu, Chun-Xia Wang, Seiichi Nishizawa, Norio Teramae

CHEMICAL COMMUNICATIONS　50　(5)　515-517　2014年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/c3cc47717g 　

ISSN：1359-7345

eISSN：1364-548X

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A series of abasic site-binding ligands conjugated with cyanine dyes have been developed for "off-on" fluorescence sensing of an orphan nucleobase in DNA duplexes and DNA-RNA hybrids.
The effect of LNA nucleobases as enhancers for the binding of amiloride to an abasic site in DNA/DNA and DNA/RNA duplexes 査読有り

Yusuke Sato, Tetsushi Sato, Takaya Sato, Seiichi Nishizawa, Norio Teramae

ORGANIC & BIOMOLECULAR CHEMISTRY　12　(37)　7250-7256　2014年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/c4ob00977k 　

ISSN：1477-0520

eISSN：1477-0539

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We report on a significant effect of locked nucleic acid (LNA) nucleobases on the binding of amiloride for abasic site (AP)-containing DNA duplexes. Fluorescence titration experiments showed that the binding affinity of amiloride for the target thymine (T) opposite an AP site significantly improves for the DNA duplexes possessing LNA nucleobases that flank the AP site, compared to the corresponding normal DNA duplexes. In particular, LNA flanking nucleobases on both 5'- and 3'-sides of the AP site are found to be effective for the enhancement of the binding affinity. From thermodynamic characterization of the amiloride binding, the loss in the binding entropy is remarkably reduced for the LNA-containing DNA duplexes, which is indeed responsible for the enhanced affinity of amiloride. Moreover, such an effect of LNA nucleobases was also observed for amiloride binding to DNA/RNA hybrid duplexes.
Rational Design for Cooperative Recognition of Specific Nucleobases Using β-Cyclodextrin-Modified DNAs and Fluorescent Ligands on DNA and RNA Scaffolds 査読有り

Akika Futamura, Asuka Uemura, Takeshi Imoto, Yusuke Kitamura, Hirotaka Matsuura, Chun-Xia Wang, Toshiki Ichihashi, Yusuke Sato, Norio Teramae, Seiichi Nishizawa, Toshihiro Ihara

Chemistry-An European Journal　19　(32)　10526-10535　2013年8月
出版者・発行元：None
DOI： 10.1002/chem.201300985 　

ISSN：0947-6539

eISSN：1521-3765
Competitive Binding of Abasic Site-Binding Ligands and Masking Ligands to DNA Duplexes for the Analysis of Single-Base Mutation 査読有り

Yusuke Sato, Tomoe Kageyama, Seiichi Nishizawa, Norio Teramae

ANALYTICAL SCIENCES　29　(1)　15-19　2013年1月
出版者・発行元：JAPAN SOC ANALYTICAL CHEMISTRY
DOI： 10.2116/analsci.29.15 　

ISSN：0910-6340

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We describe a competitive binding assay for the analysis of single-base mutation by using abasic site (AP site)-specific binding fluorescent ligands and masking ligands bound to the AP site in DNA duplexes. 2-Amino-5,6,7-trimethy1-1,8-naphthyridine (ATMND) can strongly bind to not only cytosine (C), but also thymine (T) opposite an AP site in the DNA duplexes (K-11/10(7) M-1: C, 15; T, 7.0). By contrast, in the presence of lumichrome (Lch) as a masking ligand, ATMND shows a clear binding selectivity for C over T (K-11/10(7) M-1: C, 7.0; T, 0.57), which is ascribed to the effective masking effect of Lch to the ATMND-T interaction in the AP site-containing DNA duplexes. It was shown that the competitive binding of ATMND and Lch to the AP site allowed the highly selective detection of C-related single-base mutation in DNAs amplified by polymerase chain reaction.
2,4-Diamino-6,7-dimethylpteridine as a fluorescent ligand for binding and sensing an orphan cytosine in RNA duplexes 査読有り

Yusuke Sato, Yu Toriyabe, Seiichi Nishizawa, Norio Teramae

CHEMICAL COMMUNICATIONS　49　(85)　9983-9985　2013年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/c3cc46085a 　

ISSN：1359-7345

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We report on a new fluorescent ligand, 2,4-diamino-6,7-dimethylpteridine, that can strongly and selectively bind to an orphan cytosine opposite an abasic site in RNA duplexes. We describe a significant effect of the substituents attached to the pteridine ring on the binding behavior in comparison with a structurally-similar pteridine derivative.
Competitive Assay for Theophylline Based on an Abasic Site-Containing DNA Duplex Aptamer and a Fluorescent Ligand 査読有り

Yusuke Sato, Yushuang Zhang, Seiichi Nishizawa, Takehiro Seino, Kodai Nakamura, Minjie Li, Norio Teramae

CHEMISTRY-A EUROPEAN JOURNAL　18　(40)　12719-12724　2012年10月
出版者・発行元：WILEY-V C H VERLAG GMBH
DOI： 10.1002/chem.201201298 　

ISSN：0947-6539

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A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site-containing DNA duplex aptamer (AP aptamer) in combination with an abasic site-binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23-mer AP aptamer (5'-TCT GCG TCC AG (X) under bar GCA ACG CAC AC-3'/5'-GTG TGC GTT GC (C) under bar CTG GAC GCA GA-3'; (X) under bar: the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 mu m. Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.
Ratiometric Fluorescent Signaling of Small Molecule, Environmentally Sensitive Dye Conjugates for Detecting Single-Base Mutations in DNA 査読有り

Chun-xia Wang, Yusuke Sato, Megumi Kudo, Seiichi Nishizawa, Norio Teramae

CHEMISTRY-A EUROPEAN JOURNAL　18　(31)　9481-9484　2012年7月
出版者・発行元：WILEY-V C H VERLAG GMBH
DOI： 10.1002/chem.201200219 　

ISSN：0947-6539
Base Pairing at the Abasic Site in DNA Duplexes and Its Application in Adenosine Aptasensors 査読有り

Yuanfeng Pang, Zhiai Xu, Yusuke Sato, Seiichi Nishizawa, Norio Teramae

CHEMBIOCHEM　13　(3)　436-442　2012年2月
出版者・発行元：WILEY-BLACKWELL
DOI： 10.1002/cbic.201100666 　

ISSN：1439-4227

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The binding of nucleosides to abasic site (AP site)-containing DNA duplexes (AP-DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (Kd=14.1 mu M for adenosine and 41.8 mu M for guanosine) for binding to the AP-DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP-DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP-DNAs, as revealed by ITC measurements. Next, to apply an AP-DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP-site-binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP-DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP-DNAs can serve as a new class of aptasensorsa limit of detection of 0.7 mu M was obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost-effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.
Highly selective binding of naphthyridine with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes 査読有り

Yusuke Sato, Yushuang Zhang, Takehiro Seino, Takashi Sugimoto, Seiichi Nishizawa, Norio Teramae

ORGANIC & BIOMOLECULAR CHEMISTRY　10　(20)　4003-4006　2012年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/c2ob25513h 　

ISSN：1477-0520

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We report on highly selective binding of a naphthyridine derivative with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes; the binding-induced fluorescence quenching is applicable to the analysis of a C-related single-base mutation in DNAs amplified by PCR.
Strong and Selective Binding of Amiloride to an Abasic Site in RNA Duplexes: Thermodynamic Characterization and MicroRNA Detection 査読有り

Yusuke Sato, Toshiki Ichihashi, Seiichi Nishizawa, Norio Teramae

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION　51　(26)　6369-6372　2012年
出版者・発行元：WILEY-V C H VERLAG GMBH
DOI： 10.1002/anie.201201790 　

ISSN：1433-7851
Label-Free Molecular Beacon System Based on DNAs Containing Abasic Sites and Fluorescent Ligands That Bind Abasic Sites 査読有り

Yusuke Sato, Seiichi Nishizawa, Norio Teramae

CHEMISTRY-A EUROPEAN JOURNAL　17　(41)　11650-11656　2011年10月
出版者・発行元：WILEY-BLACKWELL
DOI： 10.1002/chem.201100384 　

ISSN：0947-6539

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A new class of label-free molecular beacon (MB) system based on DNA strands that contain abasic (AP) sites (AP-DNA) and adopt stem-loop structures, in combination with fluorescent ligands that bind these AP sites, has been developed. Unlike a conventional MB, which requires covalent labeling of the MB with a fluorophore and a quencher, the developed system (APMB) does not require covalent attachment of signal transduction units. Detailed sensing functions of a series of APMB systems were examined with the aid of the fluorescent ligand named ATMND to provide insight into the design strategy for APMB systems. The effects of the stem length and the position of the AP site in the stem moiety on the fluorescence response of the APMB system were examined. Genotyping of a G/C SNP of PCR amplification products was successfully demonstrated with the APMB system and blue-fluorescent ATMND as a ligand. The APMB system was further extended to a system that utilized green-fluorescent lumiflavin.
Fluorescent aptasensors based on conformational adaptability of abasic site-containing aptamers in combination with abasic site-binding ligands 査読有り

Zhiai Xu, Yusuke Sato, Seiichi Nishizawa, Norio Teramae

BIOSENSORS & BIOELECTRONICS　26　(12)　4733-4738　2011年8月
出版者・発行元：ELSEVIER ADVANCED TECHNOLOGY
DOI： 10.1016/j.bios.2011.05.051 　

ISSN：0956-5663

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Aptamers are nucleic acids that can selectively bind to a variety of targets. Aptamers usually undergo conformational transitions from a flexible or disordered structure into a rigid or ordered structure upon target-binding. This study describes a detection method for L-argininamide (L-Arm) and adenosine based on the conformational adaptability of nucleic acid aptamers. An abasic site (AP site) was formed in the stem and close to the target-binding site of a stem-loop aptamer as an anchoring pocket for a fluorescent ligand. 3,5-Diamino-6-chloro-2-pyrazine carbonitrile (DCPC), which can bind to AP site-containing DNA duplexes by pseudo-base pairing, was utilized as a signaling reporter for the target-binding. The binding of a target to an aptamer induces the tight pairing of bases flanking the AP site, so that DCPC can effectively bind to the stem. The binding of DCPC is accompanied by a significant enhancement of its fluorescence. This new sensing method without an antisense DNA strand was demonstrated by using L-Arm and its aptamer as a model. It was confirmed that the method can sensitively detect L-Arm with a detection limit of 2.1 mu M. The proposed method was also applied to adenosine detection, where the reported sequence of an adenosine aptamer was slightly modified. The method based on an AP site-containing aptamer and an AP site-binding ligand was applicable to detection of a target in horse serum. (C) 2011 Elsevier B.V. All rights reserved.
Fluorescent trimethyl-substituted naphthyridine as a ligand for C-C mismatch detection in CCG trinucleotide repeats 査読有り

Yusuke Sato, Atsuko Honjo, Daisuke Ishikawa, Seiichi Nishizawa, Norio Teramae

CHEMICAL COMMUNICATIONS　47　(20)　5885-5887　2011年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/c1cc11370d 　

ISSN：1359-7345

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We report on the selective binding of 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) to C-C mismatch present in the hairpin structures of (CCG)(n) trinucleotide repeats that are associated with neurological diseases; this binding is accompanied by significant fluorescence quenching of ATMND.
Enhancement in fluorescence response by a quencher for amiloride upon binding to thymine opposite an abasic site in a DNA duplex 査読有り

Yusuke Sato, Jing Tian, Toshiki Ichihashi, Yuka Chinda, Zhiai Xu, Yuanfeng Pang, Seiichi Nishizawa, Norio Teramae

ANALYTICA CHIMICA ACTA　675　(1)　49-52　2010年8月
出版者・発行元：ELSEVIER SCIENCE BV
DOI： 10.1016/j.aca.2010.06.042 　

ISSN：0003-2670

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By using iodide (I(-)) as a quencher, we successfully improve the fluorescence response of amiloride when binding to thymine opposite an AP site in a 21-meric DNA duplex. From fluorescence measurements, as compared to the NaCl solutions, the addition of NaI as a quencher as well as salt to adjust the ionic strength effectively suppresses the background fluorescence from unbound amiloride in a solution. The Stern-Volmer analysis shows that the bound amiloride to the nucleobase at the AP site is unexposed to NaI quencher. Therefore the high signal-to-background fluorescence response of amiloride is obtained. Such enhancement in fluorescence response of amiloride by using the quencher can provide the significant improvement of the detection limit for DNA duplexes carrying T target base. The method presented in this study is simple and effective. The present method could be applicable to other detection system where microenvironment of fluorophores changes at a recognition event. (C) 2010 Elsevier B.V. All rights reserved.
Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride 査読有り

Arivazhagan Rajendran, Chunxia Zhao, Burki Rajendar, Viruthachalam Thiagarajan, Yusuke Sato, Seiichi Nishizawa, Norio Teramae

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS　1800　(6)　599-610　2010年6月
出版者・発行元：ELSEVIER SCIENCE BV
DOI： 10.1016/j.bbagen.2010.03.007 　

ISSN：0304-4165

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Background: We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA. Methods: The combination of experimental and theoretical methods was used. Results: The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by (31)P NMR experiments. The thermodynamics of the ligand-nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory. Conclusions: Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling. General significance: The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein-DNA interactions. However, these are not clear for the DNA-small molecule interactions and we believe that our studies will bring a new insight into such phenomena. (C) 2010 Elsevier B.V. All rights reserved.
Effect of substituents of alloxazine derivatives on the selectivity and affinity for adenine in AP-site-containing DNA duplexes 査読有り

Burki Rajendar, Arivazhagan Rajendran, Zhiqiang Ye, Eriko Kanai, Yusuke Sato, Seiichi Nishizawa, Marek Sikorski, Norio Teramae

ORGANIC & BIOMOLECULAR CHEMISTRY　8　(21)　4949-4959　2010年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/c0ob00057d 　

ISSN：1477-0520

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Using the DNA duplex containing an AP site (5'-TCC AGX GCA AC-3'/3'-AGG TCN CGT TG-5', X = AP site, N = A, T, C, or G), we have found that 2-amino-4-hydroxypteridine (pterin) selectively binds to guanine (G), and that the enhanced binding affinity for G is obtained by its methylated derivative 2-amino-6,7-dimethyl-4-hydroxypteridine (diMe pteridine). Similarly, among the cytosine (C)-selective ligands, i.e. derivatives of 2-amino-1,8-naphthyridine, a trimethyl-substituted derivative (2-amino-5,6,7-trimethyl-1,8-naphthyridine) selectively binds to C with a strong binding affinity of 1.9 x 10(7) M(-1). In the case of lumazine derivatives, pteridine-2,4(1H,3H)-dione (lumazine) binds to adenine (A), and its methylated derivative, 6,7-dimethylpteridine-2,4(1H, 3H)-dione (diMe lumazine) strongly binds to A with enhanced binding affinity, keeping the same base-selectivity. On the other hand, the benzo-annelated (with phenyl ring, 2.4 angstrom) derivative of lumazine, benzo[g] pteridine-2,4(1H, 3H)-dione (alloxazine), can bind to A selectively, whereas its methylated ligand, 7,8-dimethylbenzo[g] pteridine-,4( 1H, 3H)-dione (lumichrome) selectively binds to thymine (T) over A, C and G. Methyl-substituted lumichrome derivatives show moderate binding affinities for target nucleobases. The changes in the base-selectivity and binding affinities are discussed in detail with respect to the substituents of these ligands, considering hydrogen-bonding patterns, size of AP site and stacking interactions.
Abasic site-based DNA aptamers for analytical applications 招待有り査読有り

Seiichi Nishizawa, Yusuke Sato, Zhiai Xu, Kotaro Morita, Minjie Li, Norio Teramae

SUPRAMOLECULAR CHEMISTRY　22　(7-8)　467-476　2010年
出版者・発行元：TAYLOR & FRANCIS LTD
DOI： 10.1080/10610278.2010.484865 　

ISSN：1061-0278

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We here describe a class of duplex DNA aptamers that possess an abasic site as an active cavity for binding events. A structurally optimised 23-meric duplex (5'-TCT GCG TCC AGX GCA ACG CAC AC-3'/3'-AGA CGC AGG TCT CGT TGC GTG TG-5', X=abasic site; a propyl linker, T=receptor base) binds to riboflavin with a dissociation constant of 1.9M (at 20 degrees C, pH 7.0, I=0.11M), and it exhibits a high selectivity for riboflavin over flavin mononucleotide and flavin adenine dinucleotide. A fluorescent signalling aptamer is also developed by the incorporation of fluorescent 2-aminopurine (2-AP) into the duplex to flank the abasic site. This 2-AP-modified abasic site-containing duplex DNA (5'-TCTGC GTCCT PXT TAACG CACAC-3'/3'-AGACG CAGGA TCA ATTGC GTGTG-5', P=2-AP, X=abasic site; a propyl linker, C=receptor base) is capable of selectively binding to the bronchodilator theophylline with a dissociation constant of 10M (at 5 degrees C, pH 7.0, I=0.11M), and it is applicable to monitoring serum theophylline concentrations. In addition, we describe a design strategy for label-free aptamer-based fluorescence-signalling systems based on an abasic site-binding fluorescent ligand. A DNA aptamer against adenosine is examined as a model system, and an abasic site is designed to be incorporated into the aptamer system, so that the adenosine binding causes either a release or binding of abasic site-binding ligands with a clear fluorescent signalling. The designed system is highly selective and sensitive with a detection limit of 1-2M for adenosine. These results are discussed as a potential basis for the further development of an aptamer-based analytical assay.
Signal-Off and Signal-On Design for a Label-Free Aptasensor Based on Target-Induced Self-Assembly and Abasic-Site-Binding Ligands 査読有り

Zhiai Xu, Yusuke Sato, Seiichi Nishizawa, Norio Teramae

CHEMISTRY-A EUROPEAN JOURNAL　15　(40)　10375-10378　2009年
出版者・発行元：WILEY-V C H VERLAG GMBH
DOI： 10.1002/chem.200901226 　

ISSN：0947-6539
Label-free aptamer-based sensor using abasic site-containing DNA and a nucleobase-specific fluorescent ligand 査読有り

Zhiai Xu, Kotaro Morita, Yusuke Sato, Qing Dai, Seiichi Nishizawa, Norio Teramae

CHEMICAL COMMUNICATIONS　(42)　6445-6447　2009年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/b908345f 　

ISSN：1359-7345

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A label-free adenosine sensor with emissive response is designed based on an AP site-containing aptamer/DNA duplex and a small fluorescent molecule 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND).
Improvement of base selectivity and binding affinity by controlling hydrogen bonding motifs between nucleobases and isoxanthopterin: Application to the detection of T/C mutation 査読有り

Burki Rajendar, Yusuke Sato, Seiichi Nishizawa, Norio Teramae

BIOORGANIC & MEDICINAL CHEMISTRY LETTERS　17　(13)　3682-3685　2007年7月
出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD
DOI： 10.1016/j.bmcl.2007.04.033 　

ISSN：0960-894X

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At an abasic site in an oligo-DNA duplex, isoxanthopterin (IX) (dagger)can bind to thymine (T) and cytosine (C) with strong affinity compared to adenine and guanine, but the base selectivity for T against C is moderate. In order to improve both binding affinity and base selectivity for T against C, a methyl group is introduced to IX, which is known as 3-methyl isoxanthopterin (3-MIX),(dagger) by which binding affinity for C is expected to decrease. Indeed, 3-MIX specifically binds to T more strongly than IX and loses its binding affinity for C. The improved binding ability of 3-MIX for T would be suitable for the practical use in SNP typing related to T. (c) 2007 Elsevier Ltd. All rights reserved.
Enhancement of the binding ability of a ligand for nucleobase recognition by introducing a methyl group 査読有り

Q Dai, CY Xu, Y Sato, K Yoshimoto, S Nishizawa, N Teramae

ANALYTICAL SCIENCES　22　(2)　201-203　2006年2月
出版者・発行元：JAPAN SOC ANALYTICAL CHEMISTRY
ISSN：0910-6340

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The recognition ability of pteridine derivatives for nucleobases opposite an abasic (AP) site in an oligodeoxynucleotide (ODN) duplex is enhanced by using a propylene residue (Spacer-C3) as an AP site. The recognition ability is further enhanced both by attaching methyl groups to a fluorescent ligand and by measuring the fluorescence response at 5 degrees C; 6.2 x 10(6) M-1 of the binding constant is attained between 2-amino-6,7-dimethyl-4-hydroxypteri dine and guanine opposite the AP site in water.
Electrochemical SNPs detection using an abasic site-containing DNA on a gold electrode 査読有り

Kotaro Morita, N. B. Sankaran, Weimin Huang, Takehiro Seino, Yusuke Sato, Seiichi Nishizawa, Norio Teramae

CHEMICAL COMMUNICATIONS　(22)　2376-2378　2006年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/b517391d 　

ISSN：1359-7345

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An abasic site-containing DNA combined with lumiflavin allows amperometric determination of single nucleotide polymorphism through hydrogen bond-mediated nucleobase recognition in water by using abasic sites as a molecular recognition field.

︎全件表示 ︎最初の5件までを表示

MISC 18

Triplex-Forming Peptide Nucleic Acid Probe Having Thiazole Orange as a Base Surrogate for Fluorescence Sensing of Double-stranded RNA (vol 138, pg 9397, 2016)

Takaya Sato, Yusuke Sato, Seiichi Nishizawa

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY　139　(9)　3567-3567　2017年3月
出版者・発行元：AMER CHEMICAL SOC
DOI： 10.1021/jacs.7b01636 　

ISSN：0002-7863
RNA二重鎖を読み解く新技術－三重鎖核酸形成を利用する蛍光プローブ

佐藤貴哉, 佐藤雄介, 西澤精一

月刊「化学」　72　(4)　47-52　2017年
出版者・発行元：化学同人
ISSN：0451-1964
RNA解析・イメージングのための蛍光性核酸プローブの分子設計

佐藤貴哉, 佐藤雄介, 西澤精一

ぶんせき　2017　(10)　459-463　2017年
出版者・発行元：日本分析化学会
ISSN：0386-2178
Recent Progress in Abasic Site-binding Small Molecules for Detecting Single-base Mutations in DNA

Seiichi Nishizawa, Yusuke Sato, Norio Teramae

Analytical Sciences　30　(1)　137-142　2014年1月10日
出版者・発行元：Springer Science and Business Media LLC
DOI： 10.2116/analsci.30.137 　

ISSN：0910-6340

eISSN：1348-2246

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This review addresses our recent efforts to design AP site-binding small ligands for SNP (single-nucleotide polymorphisms) typing. First, we present a surface plasmon resonance (SPR) biosensor carrying a derivative of 3,5-diaminopyrazines. Comparison with a bulk assay based on 3,5-diaminopyrazines-DNA binding shows that the immobilization of 3,5-diaminopyrazines on the SPR sensor allows a more sensitive detection of the target DNA, and binding selectivity can be tuned by controlling salt concentrations of the sample solutions. We also present a ratiometric fluorescent probe, in which an environmentally sensitive fluorescent dye, a benzofurazan derivative, is linked through an alkyl spacer to a 2-amino-1,8-naphthyridine derivative. The binding and sensing properties of these systems are discussed as a basis for the advanced design of DNA-binding small molecules for gene detection.
シクロデキストリン修飾DNA複合体の協同的塩基認識デザイン

二村朱香, 伊本剛, 北村裕介, 佐藤雄介, 西澤精一, 井原敏博

シクロデキストリンシンポジウム講演要旨集　30th　184-185　2013年8月27日
核酸分析を指向した協同的塩基認識プログラム

二村朱香, 伊本剛, 北村裕介, 西澤精一, 佐藤雄介, 寺前紀夫, 井原敏博

分析化学討論会講演要旨集　72nd　6　2012年5月5日
核酸を足場とした特異的塩基認識機構のプログラミング

二村朱香, 伊本剛, 北村裕介, 城昭典, 西澤精一, 佐藤雄介, 寺前紀夫, 井原敏博

日本化学会講演予稿集　92nd　(3)　1047　2012年3月9日

ISSN：0285-7626
Influence of substituent modifications on the binding of 2-amino-1,8-naphthyridines to cytosine opposite an AP site in DNA duplexes: thermodynamic characterization

Yusuke Sato, Seiichi Nishizawa, Keitaro Yoshimoto, Takehiro Seino, Toshiki Ichihashi, Kotaro Morita, Norio Teramae

NUCLEIC ACIDS RESEARCH　37　(5)　1411-1422　2009年4月
出版者・発行元：OXFORD UNIV PRESS
DOI： 10.1093/nar/gkn1079 　

ISSN：0305-1048

eISSN：1362-4962

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Here, we report on a significant effect of substitutions on the binding affinity of a series of 2-amino-1,8-naphthyridines, i.e., 2-amino-1,8-naphthyridine (AND), 2-amino-7-methyl-1,8-naphthyridine (AMND), 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) and 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), all of which can bind to cytosine opposite an AP site in DNA duplexes. Fluorescence titration experiments show that the binding affinity for cytosine is effectively enhanced by the introduction of methyl groups to the naphthyridine ring, and the 1:1 binding constant (10(6) M(1)) follows in the order of AND (0.30) AMND (2.7) ADMND (6.1) ATMND (19) in solutions containing 110 mM Na (pH 7.0, at 20C). The thermodynamic parameters obtained by isothermal titration calorimetry experiments indicate that the introduction of methyl groups effectively reduces the loss of binding entropy, which is indeed responsible for the increase in the binding affinity. The heat capacity change (C(p)), as determined from temperature dependence of the binding enthalpy, is found to be significantly different between AND (161 cal/mol K) and ATMND (217 cal/mol K). The hydrophobic contribution appears to be a key force to explain the observed effect of substitutions on the binding affinity when the observed binding free energy (G(obs)) is dissected into its component terms.
Small-Molecule Binding at an Abasic Site of DNA: Strong Binding of Lumiflavin for Improved Recognition of Thymine-Related Single Nucleotide Polymorphisms

N. B. Sankaran, Yusuke Sato, Fuyuki Sato, Burki Rajendar, Kotaro Morita, Takehiro Seino, Seiichi Nishizawa, Norio Teramae

JOURNAL OF PHYSICAL CHEMISTRY B　113　(5)　1522-1529　2009年2月
出版者・発行元：AMER CHEMICAL SOC
DOI： 10.1021/jp808576t 　

ISSN：1520-6106

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The binding behavior of lumiflavin, a biologically vital ligand, with DNA duplexes containing an abasic (AP) site and various target nucleobases opposite the AP site is studied. Lumiflavin binds selectively to thymine (T) opposite the AP site in a DNA duplex over other nucleobases. Using (1)H NMR spectroscopy and fluorescence measurements, we show that ligand-DNA complexation takes place by hydrogen-bond formation between the ligand and the target nucleobases and by stacking interactions between the ligand and the nucleobases flanking the AP site. From isothermal titration calorimetric experiments, we find that ligand incorporation into the AP sites is primarily enthalpy-driven. Examination of ionic strength dependency of ligand binding with DNA reveals that ligand-DNA complexation is a manifestation of both electrostatic and nonelectrostatic interactions and that the contribution from the nonelectrolyte effect is fundamental for the stabilization of the ligand-DNA complex. In comparison to riboflavin, reported previously as a T-selective ligand, lumiflavin binds to the DNA much more strongly and is a more promising ligand for efficient detection of T-related single nucleotide polymorphisms.
2-Aminopurine-Modified Abasic-Site-Containing Duplex DNA for Highly Selective Detection of Theophylline

Minjie Li, Yusuke Sato, Seiichi Nishizawa, Takehiro Seino, Kodai Nakamura, Norio Teramae

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY　131　(7)　2448-+　2009年2月
出版者・発行元：AMER CHEMICAL SOC
DOI： 10.1021/ja8095625 　

ISSN：0002-7863

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2-Aminopurine-modified abasic-site-containing duplex [DNA 5'-TCTGC GTCCT (PX) under barT TAACG CACAC-3'/3'-AGACG CAGGA T (C) under barA ATTGC GTGTG-5'; (P) under bar = 2-aminopurine, (X) under bar = abasic site (Spacer-C3), (C) under bar = receptor base] is capable of selectively binding to the bronchodilator theophylline with a dissociation constant of 10 mu M (5 degrees C, pH 7.0, I = 0.11 M) and is applicable to monitoring serum theophylline concentrations.
Effect of methyl substitution in a ligand on the selectivity and binding affinity for a nucleobase: A case study with isoxanthopterin and its derivatives

Burki Rajendar, Arivazhagan Rajendran, Yusuke Sato, Seiichi Nishizawa, Norio Teramae

BIOORGANIC & MEDICINAL CHEMISTRY　17　(1)　351-359　2009年1月
出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD
DOI： 10.1016/j.bmc.2008.10.062 　

ISSN：0968-0896

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Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX. This leads to the known ligands 3-methyl isoxanthopterin (3-MIX) and 8-methyl isoxanthopterin (8-MIX), and the binding affinity for C or T is expected to be tuned and improved by methyl substitution. Indeed, 3-MIX selectively binds to T more strongly than IX with a binding constant of 1.5 x 10(6) M (1) and it loses its binding affinity for C. In contrast, 8-MIX selectively binds to C over T with a binding constant of 1.0 x 10(6) M (1) and the binding affinity is greatly improved compared to the parent ligand IX. The thermodynamics of the ligand-nucleotide interaction is analyzed by isothermal calorimetric titrations, and the results show that the interaction follows a 1: 1 stoichiometry and is enthalpy-driven. The introduction of methyl groups at both N-3 and N-8 positions results in an increase in enthalpy of the ligand-nucleotide interaction, which leads to the improved binding affinity. (C) 2008 Elsevier Ltd. All rights reserved.
Fluorescence and electrochemical detection of pyrimidine/purine transversion by a ferrocenyl aminonaphthyridine derivative

Kotaro Morita, Yusuke Sato, Takehiro Seino, Seiichi Nishizawa, Norio Teramae

ORGANIC & BIOMOLECULAR CHEMISTRY　6　(2)　266-268　2008年
出版者・発行元：ROYAL SOC CHEMISTRY
DOI： 10.1039/b716682f 　

ISSN：1477-0520

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A novel hydrogen bond-forming ligand for pyrimidine/purine transversion, which contains both a fluorescent naphthyridine moiety and a ferrocenyl group as an electrochemical indicator, is described. Hydrogen bond-mediated recognition for a target nucleobase at an abasic site in a DNA duplex is confirmed by both fluorescence and electrochemical measurements. The analysis by fluorescence titration reveals that the ligand shows significant fluorescent quenching upon formation of a 1 : 1 complex with the target nucleobase opposite the abasic site, and the selectivity is in the order of cytosine > thymine > adenine, guanine, reflecting the stability of the hydrogen bond formation.
Development of label-free molecular beacons based on abasic sitebinding fluorescence molecules

Yusuke Sato, Seiichi Nishizawa, Norio Teramae

Nucleic Acids Symp. Ser.　52　(46)　121-122　2008年

DOI： 10.1093/nass/nrn062 　
Competitive binding of small ligands to nucleobases in AP site-containing DNA duplexes

Tomoe Kageyama, Yusuke Sato, Seiichi Nishizawa, Norio Teramae

Nucleic Acids Symp. Ser.　52　(46)　119-120　2008年

DOI： 10.1093/nass/nrn061 　
Effect of an alkyl amino group on the binding of 1,8-naphthyridines to AP site-containing DNA duplexes

Toshiki Ichihashi, Yusuke Sato, Takehiro Seino, Seiichi Nishizawa, Norio Teramae

Nucleic Acids Symp. Ser.　52　(46)　117-118　2008年

DOI： 10.1093/nass/nrn060 　
Electrochamical and fluorescence detection of cytosine-related SNPs using ferrocenyl naphthyridine derivative

Kotaro Morita, Yusuke Sato, Takehiro Seino, Seiichi Nishizawa, Norio Teramae

Nucleic Acids Symp. Ser.　51　295-296　2007年
Strong binding of naphthyridine derivatives to cytosine in an AP site-containing DNA duplex and their application to fluorescence detection of single nucleotide polymorphisms

Yusuke Sato, Takehiro Seino, Seiichi Nishizawa, Norio Teramae

Nucleic Acids Symp. Ser.　51　313-314　2007年
Thermodynamic characterization of the binding of naphthyridines to AP site-containing DNA duplexes

Yusuke Sato, Takehiro Seino, Seiichi Nishizawa, Norio Teramae

Nucleic Acids Symp. Ser.　50　219-220　2006年

︎全件表示 ︎最初の5件までを表示

講演・口頭発表等 22

Design of fluorescent peptide probes for exosome detection

佐藤雄介

第10回日本RNAi研究会・第5回日本細胞外小胞学会　2018年8月30日
RNA・エクソソームの構造的特徴に着目した分子プローブの設計と応用招待有り

佐藤雄介

第36回九州分析化学若手の会夏季セミナー　2018年7月27日
両親媒性αヘリックスをベースとしたエクソソーム解析プローブの開発招待有り

佐藤雄介

第35回無機・分析化学コロキウム　2018年6月2日
Design of Fluorescent Probes Capable of Binding to the Specific Sites in Nucleic Acids and Their Analytical Application 招待有り

佐藤雄介

日本化学会第98春季年会　2018年3月22日
RNA 構造を識別する分子プローブの設計と応用

佐藤雄介

第21回次世代医工学研究会　2017年9月14日
Design of siRNA overhanging structure-selective fluorescent probes 国際会議

Yusuke Sato, Seiichi Nishizawa

RSC Tokyo International Conference 2017　2017年9月7日
RNA・エキソソームを認識する分析プローブの開発

佐藤雄介

平成29年度日本分析化学会東北支部若手交流会　2017年7月14日
RNA構造を識別する蛍光性ペプチド核酸プローブの開発

佐藤雄介

第77回分析化学討論会　2017年5月27日
Synthetic fluorescent probes capable of selective binding to 3’-overhanging structures in double stranded RNAs for RNA interference study

Yusuke Sato

第54回日本生物物理学会年会　2016年11月25日
細胞内siRNA解析を目指したペプチド核酸プローブの開発：カチオン性オリゴペプチド導入による機能改良

佐藤雄介, 佐藤貴哉, 金子充雅, 西澤精一

日本分析化学会年会第65年会　2016年9月14日
Design of siRNA-binding fluorescent probes for siRNA delivery analysis 国際会議

Yusuke Sato, Takaya Sato, Mitsumasa Kaneko, Seiichi Nishizawa

RNA2016　2016年6月28日
RNA構造を識別する蛍光性分子の開発

佐藤雄介

第33回無機・分析化学コロキウム　2016年6月3日
siRNAデリバリー過程解析を指向した蛍光性プローブの開発

佐藤雄介, 佐藤貴哉, 金子充雅, 西澤精一

第16回遺伝子デリバリー研究会シンポジウム　2016年5月16日
核酸特定部位に結合する蛍光性分子の開発とその分析化学的応用

日本分析化学会第63年会　2014年9月17日
蛍光性プテリジン誘導体によるRNA一塩基識別

第74回分析化学討論会　2014年5月24日
細胞内RNA解析を指向した蛍光性プローブの開発

生物物理学会東北支部会2013　2013年12月13日
細胞内siRNA解析を指向した蛍光性プローブの開発

日本分析化学会第62年会　2013年9月10日
Label-free DNA aptasensors based on abasic site-binding fluoescence molecules 国際会議

Y. Sato, Z. Xu, S. Nishizawa, N. Teramae

平成21年度日本分光学会年次講演会　2009年11月16日
Affinity labeling based on abasic site-binding fluorescent molecules 国際会議

Y. Sato, S. Nishizawa, N. Teramae

The 6th International Foroum on CHemistry of Functional Organic Chemicals Period　2009年11月15日
疎水場感受性蛍光色素を有するナフチリジン誘導体を用いた(CNG)nトリヌクレオチドリピート高感度蛍光検出

佐藤雄介, 本城温子, 石川大佑, 西沢精一, 寺前紀夫

分析化学会第58年会　2009年9月24日
ジアミノピラジン誘導体/脱塩基部位含有RNA二十差相互作用解析と小分子RNA検出

佐藤雄介, 市橋俊希, 西沢精一, 寺前紀夫

第24回生体機能関連化学シンポジウム　2009年9月13日
DNA二重鎖/リガンド相互作用を用いたアフィニティラベル化法の開発

佐藤雄介, 西沢精一, 寺前紀夫

平成21年度東日本分析若手交流会　2009年7月3日

︎全件表示 ︎最初の5件までを表示

共同研究・競争的資金等の研究課題 12

RNA標的創薬探索を指向した近赤外蛍光off-on型インジケーターの創出

佐藤雄介

提供機関：Japan Society for the Promotion of Science

制度名：Grants-in-Aid for Scientific Research

研究種目：Grant-in-Aid for Scientific Research (B)

研究機関：Tohoku University

2022年4月～ 2025年3月
表面化学構造に基づくエクソソームのサブクラス分離

久保拓也, 佐藤雄介

提供機関：Japan Society for the Promotion of Science

制度名：Grants-in-Aid for Scientific Research

研究種目：Grant-in-Aid for Challenging Research (Pioneering)

研究機関：Kyoto University

2020年7月30日～ 2023年3月31日

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本研究では，エクソソームの糖鎖機能解明を目指し，レクチン固定化SPMの作製および評価と，作製したSPMを用いたエクソソームのレクチンアフィニテークロマトグラフィー (LAC) を行った。エクソソームの化学特性を規定する条件の一つである糖鎖構造に着目し，膜表面の糖鎖構造を利用した新規分離手法の開発に着手した。Poly(ethylene-co-glycidylmethacrylate) (PEGM) を基材とするSPM (PEGM-SPM) 表面に対して，sambucus sieboldiana agglutinin (SSA) またはconcanavalin A (ConA) を導入したカラムを作製し，HPLC (High Performance Liquid Chromatography) を用いて，各カラムにおけるエクソソームの分離選択性を評価した。PEGM-SPMをテフロン素材の中空カラムに充填し，SSAまたは ConA溶液 (2 mg/mL) を封入した後，40 °Cで24 hインキュベートすることでレクチン固定化カラムを作製した。さらに，エクソソームの疎水性相互作用による非特異吸着を抑制するため，親水性ポリマーであるpolyvinylpyrrolidone で，SPMの疎水性表面をブロッキングした。作製したカラムを汎用のHPLCシステムに導入し，エクソソームに対するLAC測定を行った。その結果，エクソソームの一部がそれぞれのカラムに選択的に保持され，移動相へのhapten糖の添加によって，保持されたエクソソームを回収することが出来た。さらに回収したエクソソームについてLC-MS/MSによるプロテオーム解析を行ったところ，それぞれのカラムから溶出したエクソソームの構成タンパク質群が，大きく異なることが明らかとなった。これらの結果から，膜表面糖鎖種に基づくエクソソームのサブクラス分離に成功したことが示唆された。
蛍光プローブの結合反応に基づくエクソソーム性質解析競争的資金

佐藤雄介

2019年10月～ 2023年3月
エクソソームの構造的特徴を捉える蛍光プローブの創出とマーカーフリー解析への応用競争的資金

佐藤雄介

2019年4月～ 2021年3月
小分子RNA二重鎖イメージングを可能にする超高親和性近赤外蛍光プローブの開発競争的資金

佐藤雄介

2017年4月～ 2020年3月
ペプチド鎖を活用する超高親和性RNA結合リガンドの創成と応用

西澤精一, 佐藤雄介

提供機関：Japan Society for the Promotion of Science

制度名：Grants-in-Aid for Scientific Research

研究種目：Grant-in-Aid for Scientific Research (B)

研究機関：Tohoku University

2016年4月1日～ 2019年3月31日

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ノンコーディングRNAの高感度検出と生細胞内可視化のためのケミカルプローブとして、siRNAの細胞内デリバリー過程を可視化しうる高親和性蛍光プローブを提案した（ChemBioChem 2019）。また、二重鎖RNAの塩基配列を検出しうる三重鎖形成ペプチド核酸プローブを提案した（J. Am. Chem. Soc. 2016; Chem. Eur. J. 2017 etc）。さらに、rRNAのA-site に対する蛍光プローブを開発、阻害剤スクリーニング（FID法）における蛍光インジケーターとしての活用を提案した（Chem. Eur. J. 2018; Chem. Commun. 2019）。
高曲率膜を識別するペプチドプローブの開発とエキソソーム解析への応用競争的資金

佐藤雄介

2016年4月～ 2018年3月
ノンコーディングＲＮＡを標的とする蛍光検出リガンドの創製と応用

西澤精一, 佐藤雄介

提供機関：Japan Society for the Promotion of Science

制度名：Grants-in-Aid for Scientific Research

研究種目：Grant-in-Aid for Scientific Research (B)

研究機関：Tohoku University

2012年4月1日～ 2015年3月31日

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本研究では、ノンコーディングRNA研究に寄与しうる新しい分析手法として、ヒト免疫不全ウイルス（HIV）のtrans-activation responsive region RNA及びC型肝炎ウイルスRNAのinternal ribosome entry site（IRES）と結合しうる蛍光性リガンドを見出し、これに基づく転写阻害物質のスクリーニング法を提案した。さらに、microRNA検出のためのlight-up型の蛍光性リガンドを開発するとともに、細胞内RNA可視化のためのバイオプローブとして、small interfering RNAを標的とする蛍光プローブを開発することに成功した。
極微量小分子ＲＮＡを網羅的に解析する次世代型核酸アレイチップの開発

寺前紀夫, 山口央, 西澤精一, 佐藤雄介, 徐志愛

提供機関：Japan Society for the Promotion of Science

制度名：Grants-in-Aid for Scientific Research

研究種目：Grant-in-Aid for Scientific Research (S)

研究機関：Tohoku University

2010年4月1日～ 2015年3月31日

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小分子RNA解析を指向したリガンド開発に関しては、RNAまたはDNA/RNA二重鎖中の脱塩基部位（AP site）対面塩基（全4種類）を識別しうるリガンド構造の同定およびlight-up蛍光応答型コンジュゲートの開発に成功した。一方で、小分子RNA解析場となるナノ細孔内の特異的な特性を明らかにし、細孔内への物質移動および動態を解析しうる光導波路法を開発した。さらに、ナノ細孔内過冷却環境下でのAP site含有核酸/リガンド相互作用を用いることで、数10amol程度の標的核酸検出および一塩基識別しうる可能性を見出した。
siRNA を標的とする蛍光性リガンドの創成と細胞内解析への応用競争的資金

佐藤雄介

2012年4月～ 2014年3月
蛍光性核酸結合リガンド群を用いたマルチカラー蛍光アッセイ法の開発競争的資金

佐藤雄介

2010年4月～ 2012年3月
RNA非翻訳領域を標的とする核酸検出リガンドの創製と機能性RNA研究への応用

寺前紀夫, 西澤精一, 徐志愛, 佐藤雄介

2010年～ 2010年

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タンパク質に翻訳されずに機能するRNAは,ノンコーディングRNA (ncRNA)と呼ばれる。ここ10年程の間に,ncRNAが関与する生物現象の発見が相次ぎ,生物学の知見が新しいものに塗替えられつつある。なかでもリボスイッチは,低分子化合物との特異的結合によって遺伝子発現調節を行うユニークなncRNAで,抗生物質の新たな標的として,あるいは遺伝子発現を制御しうる技術開発に繋がる研究対象として,特に注目されているncRNAファミリーの1つである。本研究では,新たな研究領域への新たな分析ツールの提供を目指し,『ノンコーディングRNA,特にリボスイッチを標的とする蛍光性の小分子(リガンド)を開発し,これらの化合物に基づくRNA解析法並びに薬剤ハイスループットスクリーニング法を開発する』ことを目的とする。また,リボスイッチの他,『ヒト免疫不全ウイルス(HIV)のTrans-activation responsive region (TAR RNA)や16SリボソームRNAAサイト』といった遺伝子発現調節に関わるRNAの非翻訳領域を標的とする蛍光性リガンド開発も併せて試みる。本年度(平成22年4月～5月)は,ピラジン誘導体(蛍光極大波長:～400nm)が抗生物質とほぼ同等の結合親和力でTAR RNAと結合しうることを見出すとともに(K_d=770nM),蛍光性ナフチリジン誘導体(蛍光極大波長:～400nm)がアミノグリコシド類に匹敵する結合力でAサイトに結合することを見出した(K_d=530nM)。現在,より詳細な相互作用解析を進めており,それらの知見に基づいて結合機能及び蛍光応答特性の改良をさらに行うことで,ncRNAの新しい分析ツールになることが期待できる。

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