Detection of bioactive small molecules by fluorescent resonance energy transfer (FRET) in RNA-protein conjugates.
Bioactive small molecules such as metabolites and drugs play important roles in regulating biological functions. A technique for visualizing such small molecules is very useful to understand their molecular mechanisms. In this study, an RNA-protein conjugate, which consists of an RRE-RNA sensor protein (EYFP-Rev-ECFP) and an altered RRE-RNA, was constructed to detect bioactive small molecules by fluorescent resonance energy transfer (FRET). We designed a theophylline-aptamer-inserted RRE-RNA (Theo-RRE) to detect theophylline as a model target molecule. Theo-RRE formed an RNA-protein conjugate with EYFP-Rev-ECFP in the presence of theophylline. As a result, theophylline was specifically detected down to 10 microM by the FRET increase in distinction from theophylline analogue, caffeine, in cell lysates.
Endoh
Tamaki
T
Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 Minatojimaminamimachi, Chuo-ku, Kobe 650-0047, Japan. t-endoh@center.konan-u.ac.jp
Shintani
Ryo
R
Mie
Masayasu
M
Kobatake
Eiry
E
Ohtsuki
Takashi
T
Sisido
Masahiko
M
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D013806:Theophylline / Q000032:analysis*
D052157:Aptamers, Nucleotide; D011506:Proteins; D012313:RNA; D013806:Theophylline