Development real-time and quantitative RNA probe for live cell imaging

2015 Fiscal Year Final Research Report

• Project/Area Number: 26620115
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Analytical chemistry
• Research Institution: The University of Tokyo
• Principal Investigator: Yoshimura Hideaki 東京大学, 理学(系)研究科(研究院), 助教 (90464205)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: 生体分子 / 1分子計測（SMD） / バイオテクノロジー / 細胞・組織 / 分析化学

Outline of Final Research Achievements

I developed an RNA probe for dynamics analysis and quantification of target RNA in living cells. The present probe consists of circular permutated fluorescent protein (cpFP) and two RNA recognition domains. In this design, I expected that binding of RNA to the RNA recognition domains induces reversible fluorescence intensity change. The RNA recognition domain is derived from an RNA binding protein domain PUM-HD mutant that targets for mouse β-actin mRNA. I introduced this probe into cultured mouse cells and observed using a fluorescence microscope. In this observation, obvious fluorescence was detected from the cytoplasmic region. On the other hand, the ON/OFF ration of the fluorescence is not sufficient to detect single molecule target RNA in living cells. Thus I am planning to adopt dimer-type fluorescent protein to achieve high detectability of target RNA in living cells.

Free Research Field

分析化学