WEKO3
Item
Exploring and Engineering DNA/RNA Methyltransferases for Development of RNA Labeling Tools
https://doi.org/10.15102/0002000998
https://doi.org/10.15102/00020009989c19d461-4a20-4d01-a2cc-5414d661735e
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OchiaiYoshikiFulltext.pdf (18.1 MB)
Download is available from 2026/10/5.
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OchiaiYoshikiExamAbstract.pdf (64 KB)
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OchiaiYoshikiSumarry.pdf (84 KB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||||
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| PubDate | 2025-11-21 | |||||||
| Title | ||||||||
| Title | RNA標識ツールの開発に向けたDNA/RNAメチル化酵素の探索と改変 | |||||||
| Language | ja | |||||||
| Title | ||||||||
| Title | Exploring and Engineering DNA/RNA Methyltransferases for Development of RNA Labeling Tools | |||||||
| Language | en | |||||||
| Language | ||||||||
| Language | eng | |||||||
| Keyword | ||||||||
| Subject Scheme | Other | |||||||
| Subject | ancestral sequence reconstruction | biochemistry | biophysics | |||||||
| Resource Type | ||||||||
| Resource Type Identifier | http://purl.org/coar/resource_type/c_db06 | |||||||
| Resource Type | doctoral thesis | |||||||
| Identifier Registration | ||||||||
| Identifier Registration | 10.15102/0002000998 | |||||||
| Identifier Registration Type | JaLC | |||||||
| Access Right | ||||||||
| Access Rights | open access | |||||||
| Access Rights URI | http://purl.org/coar/access_right/c_abf2 | |||||||
| Author |
落合 佳樹
× 落合 佳樹
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| Author |
Ochiai, Yoshiki
× Ochiai, Yoshiki
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| Abstract | ||||||||
| Description Type | Abstract | |||||||
| Description | RNA methyltransferases (MTases) have the potential to facilitate the development of advanced synthetic biology tools for manipulating RNA methylation states and attaching covalent tags to RNA. However, it is challenging due to limited understanding of structure-function relationships in RNA MTases. In this dissertation, I explore the sequence space of the bacterial DNA methyltransferase EcoGII (M.EcoGII), a promising target for protein engineering due to its lack of sequence specificity and its residual activity on RNA by using ancestral sequence reconstruction. I thereby created SUPREM, a non-site-specific RNA MTase with better expression, thermostability, and RNA methylation activity than M.EcoGII. The SUPREM showed improved RNA methylation activity in mammalian cells and methylated a larger number of RNA methylation than M.EcoGII. Phylogenetic and mutational analysis suggested a residue that enhances the RNA methylation activity of SUPREM. Next, to expand the scope of the M.EcoGII and SUPREM applications, I investigated DNA-selective M.EcoGII homologs, spatiotemporal control of SUPREM, and the use of synthetic cofactor. A DNA-specific homolog was identified via sequence similarity network analysis. Targeted intracellular localization of SUPREM increased methylation at specific sites. A split SUPREM variants were reconstituted to functional forms in vitro and in E. coli. Our result suggested that SUPREM needs further engineering for the use of synthetic cofactors. Collectively, this study developed SUPREM and its M.EcoGII derivative enzymes which hold promise as a versatile tool for in vivo DNA and RNA methylation. |
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| Language | en | |||||||
| Exam Date | ||||||||
| 2025-08-06 | ||||||||
| Degree Conferral Date | ||||||||
| Date Granted | 2025-09-30 | |||||||
| Degree | ||||||||
| Degree Name | Doctor of Philosophy | |||||||
| Degree Referral Number | ||||||||
| Dissertation Number | 甲第209号 | |||||||
| Degree Conferrral Institution | ||||||||
| Degree Grantor Name Identifier Scheme | kakenhi | |||||||
| Degree Grantor Name Identifier | 38005 | |||||||
| Degree Grantor Name | Okinawa Institute of Science and Technology Graduate University | |||||||
| Version Format | ||||||||
| Version Type | VoR | |||||||
| Version Type Resource | http://purl.org/coar/version/c_970fb48d4fbd8a85 | |||||||
| Copyright Information | ||||||||
| Rights | © 2025 The Author. | |||||||
