Development of amplificative detection of mRNA using RNase H and its application for drug screening
| Project/Area Number |
17K01965
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Research Field |
Chemical biology
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| Research Institution | Nihon University |
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Principal Investigator |
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Project Status |
Completed (Fiscal Year 2019)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Keywords | RNase H / DNA/RNAヘテロ二重鎖 / RNA発現解析 / RNA検出プローブ / 薬剤スクリーニング / ハイスループットスクリーニング / RNA構造変化 / モレキュラービーコン |
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| Outline of Final Research Achievements |
To detect trace amounts of cellular mRNA, amplification of signals such as recycling of probes by catalytic reaction is required. In this study, we aimed to develop a novel probe that is cleaved by cellular RNase H upon binding to target RNA, thereby resulting in accumulation of the cleavage products. This probe has a stem-loop structure with a tract of rA-rU and dA-Um base pairs. A sequence complementary to the target sequence and a clamp sequence for stem formation are added to both ends. Binding of the probe to the target sequence causes a shift of the stem base pairs to allow formations of a tract of dA-U base pairs which can be cleaved by RNase H. After the cleavage, the probe is expected to be liberated from the target RNA to allow another cycle. When the probe was added to the target sequence at a ratio of 10:1, the probe was completely cleaved, suggesting that the probe experienced turnover cycles of binding and cleavage.
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| Academic Significance and Societal Importance of the Research Achievements |
標的RNA依存的にRNase Hによる切断のスイッチをオン・オフする例はこれまでになく，独自性の高い研究成果が得られたと考えている。また，本研究は，RNase Hの基質の構造形成をコントロールすることが可能であることを示し，DNA/RNA工学やRNase Hの応用の新たな展開を切り拓くという点でも有意義であると考えている。本研究で開発したプローブをさらに改良すれば，細胞内のmRNAを簡便に検出することが可能となり，mRNAの増減を指標としたハイスループットスクリーニングが容易になる期待される。従って，創薬研究を加速することが期待され，社会的にも有意義であると考えている。
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Report
(4 results)
Research Products
(2 results)
