Directed evolution of ribozymes with novel catalytic functions
| Project/Area Number |
21F20810
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Single-year Grants |
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| Section | 外国 |
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| Review Section |
Basic Section 37030:Chemical biology-related
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
横林 洋平 沖縄科学技術大学院大学, 核酸化学・工学ユニット, 教授 (70769752)
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| Co-Investigator(Kenkyū-buntansha) |
SELLES VIDAL LARA 沖縄科学技術大学院大学, 核酸化学・工学ユニット, 外国人特別研究員
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| Project Period (FY) |
2021-07-28 – 2023-03-31
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| Project Status |
Completed (Fiscal Year 2022)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2022: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2021: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Ribozyme / In vitro evolution / Directed evolution / SELEX / 進化工学 / 核酸化学 / RNA / リボザイム |
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| Outline of Research at the Start |
本研究では、大腸菌内で様々な基質を酸化・還元するリボザイムを、人工進化により獲得することを目指す。具体的には、リボザイムによる酸化・還元に伴って生成されるNAD+もしくはNADHに依存する大腸菌株を作成する。活性を有するリボザイムを転写する大腸菌のみが生存できるため、生き残った大腸菌に含まれるリボザイム配列を解析することにより、活性を有するリボザイム配列を同定する。
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| Outline of Annual Research Achievements |
Alternative systems for the selection of ribox/combox variants able to catalyze the reduction of benzyl alcohol have been set up. The first one aims to identify in an initial step variants of the ribozyme able to bind with higher affinity to the target substrate (benzyl alcohol). The system is based on capture-SELEX, and variants identified with the system will be screened for catalytic activity in a second step using a fluorescence-based assay. Additionally, we have also identified alternative substrates likely to have a higher reactivity. These include benzyl alcohol and benzaldehyde derivatives with one or more nitro or carboxyl substituents in the aromatic ring. Iterative rounds of capture-SELEX with the different substrates are currently being performed.
The second strategy is based on the previously mentioned selection system which exploits the requirement of oxidized NAD+ for anaerobic growth (Selles Vidal, Murray and Heap, 2021). The library of combox variants, as well as wild-type combox, have been cloned into the appropriate plasmids for the selection system. Additionally, a classical protein benzyl alcohol dehydrogenase has been also cloned into the same vector. We have demonstrated that cells transformed with the protein benzyl alcohol dehydrogenase can grow anaerobically in medium supplemented with benzyl alcohol, proving that this pair of substrate and enzymatic activity are suitable for the selection system. We are currently applying the system to the RNA-based benzyl alcohol dehydrogenases.
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| Research Progress Status |
令和4年度が最終年度であるため、記入しない。
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| Strategy for Future Research Activity |
令和4年度が最終年度であるため、記入しない。
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Report
(2 results)
Research Products
(1 results)
