RNA-interacting FRET assay that allows realtime detection of RNA-protein interaction

2016 Fiscal Year Final Research Report

• Project/Area Number: 15K14554
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Plant molecular biology/Plant physiology
• Research Institution: Kyoto Prefectural University
• Principal Investigator: Obokata Junichi 京都府立大学, 生命環境科学研究科, 教授 (50185667)
• Research Collaborator: SATOH Soichirou ; MATUO Mitsuhiro ; SUGIOKA Atsushi
• Project Period (FY): 2015-04-01 – 2017-03-31
• Keywords: RNA-protein interaction / FRET / RNA-Interacting FRET / RiFRET / psbE / CREF3 / venus

Outline of Final Research Achievements

In this study, we attempted to develop a novel analytical method to detect the RNA-protein interaction by using the fluorescence resonance energy transfer (FRET), and we named this new method as RNA-interacting FRET (RiFRET). For this sake, we introduced the aptamer sequence, namely iSpinach that binds to the fluorophore DFHBI, to the given RNAs, and then examined the FRET between this fluorescent RNA molecules and fluorescently labeled RNA-binding proteins, by spectrofluorometer. In this study, our effort was focused on making the RiFRET system between chloroplast psbE RNA editing site and CREF3, a PPR protein that binds to this editing site as an RNA editing trans-factor labelled by yellow fluorescent protein, Venus. Unfortunately, we still refrain from declaring the success in detecting RiFRET effect between the above molecules. We need improvement of RiFRET system especially in stabilizing the iSpinach-DFHBI fluorescence.

Free Research Field

ゲノム生物学、分子生物学