Development of a translation system using expanded genetic alphabets through unnatural base pairs

2013 Fiscal Year Final Research Report

• Project/Area Number: 24710256
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Living organism molecular science
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: KIMOTO Michiko 独立行政法人理化学研究所, ライフサイエンス技術基盤研究センター, 上級研究員 (20415144)
• Project Period (FY): 2012-04-01 – 2014-03-31
• Keywords: 人工塩基対 / 翻訳 / 転写 / 複製 / tRNA / mRNA / T7 RNA ポリメラーゼ / 無細胞タンパク質合成系

Research Abstract

For the expansion of the genetic alphabet of DNA, we studied several codon-anticodon interactions in a cell-free translation system involving hydrophobic unnatural bases. For this project, we mainly worked on to establish efficient transcription systems and their template DNA preparation methods which allow incorporation of hydrophobic unnatural bases at desired potions into DNA and RNA. The results found from this project would open up further development on a translation system involving unnatural base pairs orthogonal to natural base pairs.

Research Products

• [Journal Article] Site-specific functional labeling of nucleic acids by in vitro replication and transcription using unnatural base pair systems (2013)
  • Author(s): M. Kimoto, Y. Hikida, I. Hirao
  • Journal Title: Israel. J. Chem Volume: 53 Pages: 450-468
  • DOI: 10.1002/ijch.20130013
  • Peer Reviewed
• [Journal Article] Generation of high-affinity DNA aptamters using an expanded genetic alphabet (2013)
  • Author(s): M. Kimoto, R. Yamashige, K. Matsunaga, S. Yokohama, I. Hirao
  • Journal Title: Nat. Biotechnol Volume: 31 Pages: 453-457
  • DOI: 10.1038/nbt.2556
  • Peer Reviewed
• [Journal Article] Natural versus artificial creation of base pairs in DNA : origin of nucleobases from the perspectives of unnatural base pair studies (2012)
  • Author(s): Hirao I, Kimoto M, Yamashige R
  • Journal Title: Acc. Chem. Res Volume: 45 Pages: 2055-65
  • DOI: 10.1021/ar200257x
  • Peer Reviewed
• [Journal Article] Site-specific functionalization of RNA molecules by an unnatural base pair transcription system via click chemistry (2012)
  • Author(s): Ishizuka T, Kimoto M, Sato A, Hirao I
  • Journal Title: Chem. Commun Volume: 48 Pages: 10835-7
  • DOI: 10.1039/c2cc36293g
  • Peer Reviewed
• [Journal Article] Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma (2012)
  • Author(s): Hirao I, Kimoto M
  • Journal Title: Proceedings of the Japan Academy, Series B Volume: 88 Pages: 345-67
  • DOI: 10.2183/pjab.88.345
  • Peer Reviewed
• [Journal Article] PCR amplification and transcription for site-specific labeling of large RNA molecules by a two-unnatural-base-pair system (2012)
  • Author(s): Kimoto M, Yamashige R, Yokoyama S, Hirao
  • Journal Title: J. Nucleic Acids Pages: 230943
  • DOI: 10.1155/2012/230943
  • Peer Reviewed
• [Presentation] Site-specific functional labeling of RNA molecules by using unnatural base pair systems (2014)
  • Author(s): Michiko Kimoto
  • Organizer: The 65th Fujihara Seminar International Symposium on Synthetic Biology of Unnatural Base Pairs and Amino Acids
  • Place of Presentation: 北海道・苫小牧
  • Year and Date: 20141001-04
• [Presentation] Thermal stability of hydrophobic unnatural base pairs in duplexes (2014)
  • Author(s): Michiko Kimoto, Ichiro, Hirao
  • Organizer: XVIth SCNAC
  • Place of Presentation: チェコ・チェスキークルムノフ
  • Year and Date: 20140608-13
• [Presentation] Variation of Genetic Alphabets of Nucleobases (2013)
  • Author(s): Michiko Kimoto, Ichiro Hirao
  • Organizer: International Astrobiology workshop 2013
  • Place of Presentation: 相模原・JAXA
  • Year and Date: 20131128-30
• [Presentation] 複製可能な人工塩基対を組み込んだDNAを創る (2013)
  • Author(s): 木本路子
  • Organizer: 「細胞を創る」研究会6
  • Place of Presentation: 慶応技術大学鶴岡タウンキャンパス
  • Year and Date: 20131114-15
• [Presentation] High-affinity DNA aptamer selection by a genetic alphabet expansion PCR system (2013)
  • Author(s): Michiko Kimoto, Ken-ichiro Matsunaga, Rie Yamashige, Ichiro Hirao
  • Organizer: 第51回日本生物物理学会年会シンポジウム
  • Place of Presentation: 国立京都国際会館
  • Year and Date: 20131028-30
• [Presentation] Expanding the Genetic Alphabet for Generating High-affinity DNA Aptmaer (2013)
  • Author(s): Michiko Kimoto, Ken-ichiro Matsunaga, Rie Yamashige, Ichiro hirao
  • Organizer: OTS Annual Meeting 2013
  • Place of Presentation: イタリア・ナポリ
  • Year and Date: 20131006-08
• [Presentation] Site-specific labeling of RNA by a replication-transcription system using a genetic alphabet expansion with two unnatural base pairs (2013)
  • Author(s): Michiko Kimoto, Rie Yamashige, Ichiro Hirao
  • Organizer: 3rd International Conference on Nucleic Acid-Protein Chemistry and Structural Biology
  • Place of Presentation: 米国・アトランタ
  • Year and Date: 20130913-15
• [Presentation] Creation of DNA aptamers using libraries with five different bases by an unnatural base pair system (2012)
  • Author(s): Michiko Kimoto, Rie Yamashige, Ken-ichiro Matsunaga, Shigeyuki Yokoyama, Ichiro Hirao
  • Organizer: 第39回国際核酸化学シンポジウム
  • Place of Presentation: 名古屋大学豊田講堂
  • Year and Date: 20121113-15
• [Presentation] The third base pair'Ds-Px'in PCR amplification (2012)
  • Author(s): Rie Yamashige, Michiko Kimoto, Yusuke Takezawa, Akira Sato, Tsuneo Mitsui, Shigeyuki Yokoyama, Ichiro Hirao
  • Organizer: XX IRT-20th International Roundtable on Nucleosides, Nucleotides and Nucleic Acids
  • Place of Presentation: カナダ、モントリオール
  • Year and Date: 20120805-09
• [Presentation] 人工塩基対システムおよびクリック反応による機能性RNAの部位特異的修飾 (2012)
  • Author(s): 石塚匠, 木本路子, 佐藤旭, 横山茂之, 平尾一郎
  • Organizer: 第14回日本RNA学会年会
  • Place of Presentation: 東北大学百周年記念会館
  • Year and Date: 20120718-20
• [Presentation] Site-specific labeling of RNA molecules by an unnatural base pair transcription system coupled with click chemistry (2012)
  • Author(s): Takumi Ishizuka, Michiko Kimoto, Akira Sato, Shigeyuki Yokoyama, Ichiro Hirao
  • Organizer: Fluorescent Biomolecules and their Building Blocks - Design and Applications
  • Place of Presentation: スウェーデンヨーテボリ
  • Year and Date: 20120705-08
• [Presentation] Site-specific fluorescent probing of RNA molecules by an unnatural base pair system and its applications (2012)
  • Author(s): Michiko Kimoto, Yasushi Hikida, Shuang Liu, Shigeyuki Yokoyama, Ichiro Hirao
  • Organizer: Fluorescent Biomolecules and their Building Blocks - Design and Applications
  • Place of Presentation: スウェーデンヨーテボリ
  • Year and Date: 20120705-08
• [Book] Creation of Unnatural Base Pair Systems Toward New DNA/RNA Biotechnologies, Chemical Biology of Nucleic Acids–Fundamentals and Clinical Applications- RNA Technologies (2014)
  • Author(s): Michiko Kimoto, Ichiro, Hirao
  • Total Pages: 131-148
  • Publisher: Springer-Verlag
• [Remarks]
  • URL: http://protein.gsc.riken.jp/hirao/