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Structural insights into lab-coevolved RNA–RBP pairs and applications of synthetic riboswitches in cell-free system 査読あり 国際誌
Keisuke Fukunaga, Takamasa Teramoto, Momoka Nakashima, Toshitaka Ohtani, Riku Katsuki, Tomoaki Matsuura, Yohei Yokobayashi, Yoshimitsu Kakuta
Nucleic Acids Research 53 ( 6 ) 2025年3月
担当区分：筆頭著者, 責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Oxford University Press
CS1–LS4 and CS2–LS12 are ultra-high affinity and orthogonal RNA–protein pairs that were identified by PD-SELEX (Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment). To investigate the molecular basis of the lab-coevolved RNA–RBP pairs, we determined the structures of the CS1–LS4 and CS2–LS12 complexes and the LS12 homodimer in an RNA-free state by X-ray crystallography. The structural analyses revealed that the lab-coevolved RNA–RBPs have acquired unique molecular recognition mechanisms, whereas the overall structures of the RNP complexes were similar to the typical kink-turn RNA-L7Ae complex. The orthogonal RNA–RBP pairs were applied to construct high-performance cell-free riboswitches that regulate translation in response to LS4 or LS12. In addition, by using the orthogonal protein-responsive switches, we generated an AND logic gate that outputs staphylococcal γ-hemolysin in cell-free system and carried out hemolysis assay and calcein leakage assay using rabbit red blood cells and artificial cells, respectively.
DOI： 10.1093/nar/gkaf212
DOI： 10.1093/nar/gkaf212
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Switchable and orthogonal gene expression control inside artificial cells by synthetic riboswitches 査読あり 国際共著 国際誌
Yuta Ishii, Keisuke Fukunaga, Aileen Cooney, Yohei Yokobayashi, Tomoaki Matsuura
Chemical Communications 60 ( 46 ) 5972 - 5975 2024年5月
担当区分：責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Chemical Communications
Here we report two novel synthetic riboswitches that respond to ASP2905 and theophylline and function in reconstituted cell-free protein synthesis (CFPS) system. We encapsulated the CFPS system as well as DNA-templated encoding reporter genes regulated by these orthogonal riboswitches inside liposomes, and achieved switchable and orthogonal control over gene expression by external stimulation with the cognate ligands.
DOI： 10.1039/d4cc00965g
DOI： 10.1039/d4cc00965g
その他リンク： https://t2r2.star.titech.ac.jp/cgi-bin/publicationinfo.cgi?q_publication_content_number=CTT100928039
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Small-Molecule Aptamer for Regulating RNA Functions in Mammalian Cells and Animals 査読あり 国際誌
Fukunaga K., Dhamodharan V., Miyahira N., Nomura Y., Mustafina K., Oosumi Y., Takayama K., Kanai A., Yokobayashi Y.
Journal of the American Chemical Society 145 ( 14 ) 7820 - 7828 2023年4月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of the American Chemical Society
Synthetic riboswitches that can regulate gene expression by a small molecule recognized by an RNA aptamer in mammalian cells have various potential applications in biotechnology and medicine. However, the variety of small molecules and their cognate aptamers that have been demonstrated to function in mammalian cells is limited. The currently available aptamer-ligand pairs also require high small molecule concentrations to enable gene regulation, making them less desirable for industrial and biomedical applications. We conducted in vitro selection of RNA aptamers against a small molecule ASP7967 whose structure is closely related to ASP2905, a known inhibitor of potassium voltage-gated channel sub-family H member 3 (KCNH3). One of the aptamers selected (AC17-4) was found to be functional in HEK293 cells, and it was used to design aptazyme-based riboswitches that can activate gene expression (>10-fold) in the presence of ASP2905 or ASP7967 at as low as 5 μM in the culture medium. An aptazyme-based riboswitch was successfully used to regulate human erythropoietin expression in mice injected with an adeno-associated virus (AAV8) vector using orally administered ASP7967. Furthermore, by combining aptazyme-based and exon-skipping riboswitch mechanisms, an ON/OFF ratio approaching 300 was achieved with a low basal expression level in cultured cells.
DOI： 10.1021/jacs.2c12332
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Directed evolution of orthogonal RNA-RBP pairs through library-vs-library in vitro selection 査読あり 国際誌
Fukunaga K., Yokobayashi Y.
Nucleic Acids Research 50 ( 2 ) 601 - 616 2022年1月
担当区分：筆頭著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Nucleic Acids Research
RNA-binding proteins (RBPs) and their RNA ligands play many critical roles in gene regulation and RNA processing in cells. They are also useful for various applications in cell biology and synthetic biology. However, re-engineering novel and orthogonal RNA-RBP pairs from natural components remains challenging while such synthetic RNA-RBP pairs could significantly expand the RNA-RBP toolbox for various applications. Here, we report a novel library-vs-library in vitro selection strategy based on Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment (PD-SELEX). Starting with pools of 1.1 × 1012 unique RNA sequences and 4.0 × 108 unique phage-displayed L7Aescaffold (LS) proteins, we selected RNA-RBP complexes through a two-step affinity purification process. After six rounds of library-vs-library selection, the selected RNAs and LS proteins were analyzed by next-generation sequencing (NGS). Further deconvolution of the enriched RNA and LS protein sequences revealed two synthetic and orthogonal RNA-RBP pairs that exhibit picomolar affinity and >4000-fold selectivity.
DOI： 10.1093/nar/gkab527
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Design of Mammalian ON-Riboswitches Based on Tandemly Fused Aptamer and Ribozyme 査読あり
Mustafina K., Fukunaga K., Yokobayashi Y.
ACS Synthetic Biology 9 ( 1 ) 19 - 25 2020年1月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：ACS Synthetic Biology
Self-cleaving ribozymes engineered to be activated or inhibited by a small molecule binding to an RNA aptamer inserted within a ribozyme (aptazymes) have proven to be useful for controlling gene expression in living cells. In mammalian cells, an aptazyme embedded in the 5′ or 3′ untranslated region of an mRNA functions as a synthetic riboswitch to chemically regulate gene expression. However, the variety of aptazyme architectures and the ribozyme scaffolds that have been used for mammalian riboswitches has been limited. In particular, fewer synthetic riboswitches that activate gene expression in response to a small molecule (ON-switches) in mammalian cells have been reported compared to OFF-switches. In this work, we developed mammalian riboswitches that function as guanine-Activated ON-switches based on a novel aptazyme architecture in which an aptamer and a ribozyme are fused in tandem. The riboswitch performance was optimized by fine-Tuning the stability of a critical stem that controls the ribozyme structure and function, yielding switches with ON/OFF ratios greater than 6.0. Our new aptazyme architecture expands the RNA device toolbox for controlling gene expression in mammalian cells.
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若手研究者からのメッセージ
福永 圭佑（ 担当： 単著）
日本化学会 バイオテクノロジー部会 ニュースレター 2022年
記述言語：日本語
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PD-SELEX法を用いた直交性RNA-RBPペアの指向性進化
福永 圭佑（ 担当： 単著）
日本化学会 生体機能関連化学部会 ニュースレター 2021年
記述言語：日本語
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Site-specific N-terminal Fluorescent Labeling of Unprotected Peptides
Keisuke Fukunaga, Takayoshi Watanabe, and Takahiro Hohsaka（ 担当： 共著）
Peptide Science 2015 2016年
記述言語：英語
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An Artificial Molecule-Peptide Hybrid Discovered via the 10BASEd-T Binds to the Substrate-Binding Site of Glutathione S-Transferase
Keisuke Fukunaga, Takaaki Hatanaka, Yuji Ito, and Masumi Taki（ 担当： 共著）
Peptide Science 2013 2014年
記述言語：英語
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Construction of a Peptide Expression Vector Library for Phenotypic Screening of Bioactive Peptides in Yeast
Keisuke Fukunaga and Masumi Taki（ 担当： 共著）
Peptide Science 2013 2014年
記述言語：英語
講演・口頭発表等 【 表示 ／ 非表示 】
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Exploration of RNA Aptamers Against Photoreceptor Protein DrBphP Through SELEX Method 国際会議
Zimu Zhang, Keisuke Fukunaga, Tomoaki Matsuura
13th ELSI International Symposium 2025
開催年月日： 2025年1月
記述言語：英語 会議種別：ポスター発表
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Searching for proper fatty acids for the construction of growing vesicles 国際会議
Ayumi Koyama, Takayoshi Watanabe, Kaito Seo, Keisuke Fukunaga, Tomoaki Matsuura
13th ELSI International Symposium 2025
開催年月日： 2025年1月
記述言語：英語 会議種別：ポスター発表
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Orthogonal gene regulation using RBP-based riboswitches inside artificial cells 国際会議
Toshitaka Ohtani, Keisuke Fukunaga, Yoshikazu Tanaka, Tomoaki Matsuura
13th ELSI International Symposium 2025
開催年月日： 2025年1月
記述言語：英語 会議種別：ポスター発表
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Investigating RNA-Phospholipid Interactions: Insights into the Mechanisms of Protocellular Assembly 国際会議
Arshjot Mann, Keisuke Fukunaga, Tomoaki Matsuura
13th ELSI International Symposium 2025
開催年月日： 2025年1月
記述言語：英語 会議種別：ポスター発表
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Molecular Threads: Unravelling the RNA-Lipid interactions in Early Cell Assembly
Arshjot Mann, Keisuke Fukunaga, Tomoaki Matsuura
Astrobiology Graduate Conference Japan (AbGradJ) 2024年12月
開催年月日： 2024年12月
記述言語：英語 会議種別：ポスター発表
Works（作品等） 【 表示 ／ 非表示 】
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Crystal structure of the L7Ae derivative protein LS12 in RNA-free state
Takamasa Teramoto, Momoka Nakshima, Keisuke Fukunaga, Yohei Yokobayashi, Yoshimitsu Kakuta
2025年4月2日
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Crystal structure of the L7Ae derivative protein LS12 in complex with its co-evolved target CS2 RNA
Takamasa Teramoto, Momoka Nakshima, Keisuke Fukunaga, Yohei Yokobayashi, Yoshimitsu Kakuta
2025年4月2日
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Crystal structure of the L7Ae derivative protein LS4 in complex with its co-evolved target CS1 RNA
Takamasa Teramoto, Momoka Nakshima, Keisuke Fukunaga, Yohei Yokobayashi, Yoshimitsu Kakuta
2025年4月2日
受賞 【 表示 ／ 非表示 】
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第13回新化学技術研究奨励賞
2024年6月 公益社団法人新化学技術推進協会（JACI）
本人
受賞区分：出版社・新聞社・財団等の賞
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支部長賞
2023年7月 日本生物工学会東日本支部
本人
受賞区分：国内学会・会議・シンポジウム等の賞
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部会講演賞
2021年9月 第15回バイオ関連化学シンポジウム
本人
受賞区分：国内学会・会議・シンポジウム等の賞
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FIBER核酸化学 若手講演賞
2021年8月 FIBER日本核酸化学会若手フォーラム
本人
受賞区分：国内学会・会議・シンポジウム等の賞
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若手口頭発表優秀賞
2013年11月 第4回 アジア−太平洋国際ペプチドシンポジウム
本人
受賞区分：国際学会・会議・シンポジウム等の賞