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哺乳類細胞で機能する人工リボスイッチの創製
https://doi.org/10.15102/1394.00001756
https://doi.org/10.15102/1394.00001756641490da-d13a-4081-a714-8355b3d1828a
名前 / ファイル | ライセンス | アクション |
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Full_Text (5.9 MB)
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Final_Exam_Abstract (42.9 kB)
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Item type | 学位論文 / Thesis or Dissertation(1) | |||||||
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公開日 | 2021-03-01 | |||||||
タイトル | ||||||||
言語 | ja | |||||||
タイトル | 哺乳類細胞で機能する人工リボスイッチの創製 | |||||||
タイトル | ||||||||
言語 | en | |||||||
タイトル | Engineering Synthetic Riboswitches for Mammalian Cells | |||||||
言語 | ||||||||
言語 | eng | |||||||
資源タイプ | ||||||||
資源タイプ識別子 | http://purl.org/coar/resource_type/c_db06 | |||||||
資源タイプ | doctoral thesis | |||||||
ID登録 | ||||||||
ID登録 | 10.15102/1394.00001756 | |||||||
ID登録タイプ | JaLC | |||||||
アクセス権 | ||||||||
アクセス権 | open access | |||||||
アクセス権URI | http://purl.org/coar/access_right/c_abf2 | |||||||
著者 (英) |
Mustafina, Kamila
× Mustafina, Kamila
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抄録 | ||||||||
内容記述タイプ | Other | |||||||
内容記述 | Riboswitches are natural and artificial noncoding RNA elements capable of controlling gene expression in response to chemical signals without direct involvement of protein factors. One strategy for engineering synthetic riboswitches is to combine an aptamer –a short RNA sequence that specifically binds to a ligand– and a self-cleaving ribozyme to create an aptazyme whose self-cleavage activity is regulated by the aptamer ligand. These aptazymes can be embedded in the 3’UTR of mRNAs to chemically control gene expression in mammalian cells. This property of riboswitches opens a wide area of applications in biology and medicine. However, engineering riboswitches that function efficiently in mammalian cells remains challenging, partly due to the difficulties associated with generating and screening aptamers and aptazymes that function in the cellular environment. In this thesis, I introduce two new ribozyme scaffolds for aptazyme engineering in mammalian cells. First, I identified highly active variants in mammalian cells from the twister and pistol ribozyme families. Then I used them as scaffolds for a new aptazyme architecture, where the aptamer is placed immediately upstream of the ribozyme in a tandem configuration. I optimized this design in mammalian cells, and then generated randomized libraries of 4096 aptazyme variants for high-throughput in vitro screening to identify switches with high on-off ratios. Although the method allowed characterization of a large number of variants, their activities were not always reproducible when tested in cells. Therefore, in addition to in vitro screening, I explored rational design approaches for the same tandem architecture. I finetuned the activity of the aptazyme by systematically varying the length of the inserted competing stem and introducing single-nucleotide mismatches and spacers. Using this method, I developed mammalian riboswitches with on-off ratios greater than 6.0 for the twister scaffold, and greater than 5.0 for the circularly permuted pistol scaffold. Lastly, learning from the experience of high-throughput in vitro screening and rational design in cells, I used high-throughput sequencing to directly screen for functional aptazymes in mammalian cells by quantifying the uncleaved fractions of aptazyme-embedded mRNAs. I verified this method with a small twister ribozyme library containing 256 variants and then applied it for a larger circularly permuted pistol ribozyme library consisting of 1024 variants. This work expands both the tools and the methods available in the field of RNA engineering. Rational and high-throughput design strategies developed in this thesis can be applied to generate other RNA devices for biomedical and synthetic biology applications. | |||||||
口頭試問日 | ||||||||
2021-01-13 | ||||||||
学位授与年月日 | ||||||||
学位授与年月日 | 2021-02-28 | |||||||
学位名 | ||||||||
学位名 | Doctor of Philosophy | |||||||
学位授与番号 | ||||||||
学位授与番号 | 甲第69号 | |||||||
学位授与機関 | ||||||||
学位授与機関識別子Scheme | kakenhi | |||||||
学位授与機関識別子 | 38005 | |||||||
学位授与機関名 | Okinawa Institute of Science and Technology Graduate University | |||||||
著者版フラグ | ||||||||
出版タイプ | VoR | |||||||
出版タイプResource | http://purl.org/coar/version/c_970fb48d4fbd8a85 | |||||||
権利 | ||||||||
権利情報 | © 2021 The Author. |