| Project/Area Number |
23KF0109
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Multi-year Fund |
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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 | The University of Tokyo |
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Principal Investigator |
菅 裕明 東京大学, 大学院理学系研究科(理学部), 教授 (00361668)
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| Co-Investigator(Kenkyū-buntansha) |
RICHAUD ALEXIS 東京大学, 大学院理学系研究科(理学部), 外国人特別研究員
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| Project Period (FY) |
2023-07-26 – 2026-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2025: ¥100,000 (Direct Cost: ¥100,000)
Fiscal Year 2024: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2023: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | Unnatural nucleotide / RNA synthesis / Ribosomal translation / Genetic code expansion |
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| Outline of Research at the Start |
In fiscal year 2023, we will focus on optimizing the ribosomal translation using UBPs as it has never been done strictly in a reproducible and efficient way. By December 2023, we hope to have a strong protocol for genetic code expansion using one extra UBP and efficient aminoacylation of the non-standard tRNA by mean of Flexizyme. Incorporation of 8 unnatural amino-acids into the system should be sufficient to generate a high impact publication. Future work will focus on the development of the modified RaPID system for the discovery of new compounds against undruggable targets.
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| Outline of Annual Research Achievements |
We synthesized non-standard mRNAGACUZP and non-standard tRNAGACUZP by PCR amplification, and T7 transcription from purchased primers. The integrity of mRNAGACUZP was controlled by gel electrophoresis (PAGE) and ribosomal translation experiment using a model peptide. To design the most efficient tRNA for our system we screened a series of modified tRNAAsnE2 and tested them for competitive translation on seven XGX different codons. We succeeded in finding three tRNAAsnE2 loaded with 4-fluorophenylalanine (4F-Phe) able to compete for the natural codon against proteinogenic amino-acid incorporation. Finally, Flexizyme was exploited to aminoacylate the desired 4F-Phe to the 3’-end of our newly designed synthetic tRNAGACUZP. Aminoacylation was confirmed by Biotin/Streptavidin complex based PAGE.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Surprisingly, translation experiment using both mRNAGACUZP and tRNAGACUZP yielded only peptides containing natural amino-acids leading the research plan towards deciphering the mechanism of ribosomal translation while using unnatural nucleotides. We are currently controlling the integrity of synthesized tRNAs using sequencing and will also control their 3D secondary structure. We are also considering testing other unnatural nucleotides more prone to be accepted by E. coli ribosome. For this purpose, we have already contacted a collaborator in Singapore who can provide primers oligomers and nucleotides.
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| Strategy for Future Research Activity |
Once the mechanism of ribosomal translation while using unnatural nucleotides is understood, we plan to select our best tRNAGACUXX and evaluate the translation efficiency through incorporation of 14C-labeled Asp, separation of the resulting products by PAGE and autoradiography. To create a novel genetic code useful for most, we need to select unnatural AAs relevant for the scientific community, accessible for tRNAGACTXX aminoacylation and translation. 6 non-proteinogenic amino-acids were already selected for the novel genetic code including 4-fluorophenylalanine and 1-methyltryptophan which has never been reported in genetic code reprogramming. Finally, we plan to test this expanded genetic code for experimental conditions used during the RaPID selection system developed by our group.
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