旭硝子財団助成研究成果報告2018

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36真壁　幸樹29Koki MAKABE吉村　英哲１生細胞内RNA定量経時追跡法の開発30HideakiYOSHIMURAタンパク質工学を駆使した自己組織化ナノシートの分子設計(2016採択)Molecular design of self-assembly mimic nano sheets based on protein engineering(Project 2016)(2016採択)Development of a method for time-course quantification of RNA in single living cells(Project 2016)旭硝子財団　助成研究成果報告（2018）本研究の大きな目標は予測可能な立体構造の設計を原子レベルの精密さで作り出すことを可能とする新規なナノマテリアル制御技術の創出である．そこで今回，申請者がこれまで取り組んできた蛋白質の自己組織化を分子内に取り込んだ人工蛋白質を用いて，その構造と機能の制御を目指した．βシートが二分子重なった人工蛋白質では，この会合の界面に存在するアミノ酸配列を変化させて，会合状態を変化させることに成功した．また，βシート表面に化学反応活性の高いチオールの導入を行い，その分子構造の決定に成功した．今後はヘテロ二量体の構築や化学修飾の導入に進み構造の制御されたナノマテリアルの構築を行いたい．The specific aim of this project is that the creation of novel protein nano materials with their three-di-mensional position of the atoms are predictably controlled with atomic resolution accuracy. To this end, here we have constructed model proteins, which were based on a model protein we have been studied for modeling the self-assembly process of protein aggregation. In the case of the beta-sheet di-mer model protein, we have achieved the control of the dimerization by introducing the amino-acid mutations on the interface. We have also successfully determined the 3D structure of thiol introduced variants. We are now working on the construction of hetero dimer construct and a chemical modifica-tion of this models.本研究はRNAの局在および動態を生細胞内で可視化するプローブの開発を目的として2種類のプローブ作成を試みた．1つは標的RNAの生細胞内1分子動態リアルタイム可視化を指向したプローブ，もう1つは標的RNAの生細胞内局在解析と定量を長時間に渡って実現するプローブである．前者においてはテロメア繰り返し配列RNAを標的として可視化を行い，生細胞内で拡散モードと静止モードの2種類の動態があることや，関連タンパク質との共局在時間と局在の可視化定量を実現した．後者においてはβアクチンmRNAを標的として，RNA存在下で蛍光性を示しRNAを分解すると蛍光性を消失する，定量解析が可能なプローブを構築した．本研究で開発したプローブ群により，時空間的に微視的・巨視的両面から生細胞内RNAの可視化解析を実現する基盤技術の礎が確立したと言える．In the present study, I developed two types of RNA probes for visualization of RNA motility and localiza-tion in living cells. One is to visualize single-molecule real-time RNA motility, and the other is to monitor quantity and localization of target RNA molecules. In the development of the former probe, I chose te-lomeric repeat-containing RNA, TERRA, as the target RNA for visualization. Through the observation of TERRA using the present probe, I found diversity of TERRA motility in living cells and colocalization with telomere-related proteins. On the later probe, I addressed beta-actin mRNA as the target, and mon-itored alteration of fluorescence intensity of the probe upon addition and decomposition of the RNA using a fluorescence spectrometer. This series of RNA probes will provide a platform to analyze RNA dynamics and function in living cells from both of micro- and macroscopic views.