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.