A Dynamic FRET Reporter of Gene Expression Improved by Functional Screening

Abstract

Here, we describe a reporter system that consists of a FRET biosensor and its corresponding aptamer. The FRET biosensor employs the synthetic aptamer binding peptide Rsg1.2 sandwiched between mutants of the Green Fluorescent Protein and undergoes FRET when binding its corresponding Rev Responsive Element (RRE) RNA aptamer. We developed a novel approach to engineer FRET biosensors by linker extension and screening to improve signal strength of the biosensor which we called VAmPIRe (Viral Aptamer binding Peptide based Indicator for RNA detection). We demonstrate that the system is quantitative, reversible and works with high specificity in vitro and in vivo in living bacteria and mammalian cells. Thus, VAmPIRe may become valuable for RNA localizations and as a dynamic RNA-based reporter for live cell imaging. Moreover, functional screening of large libraries as demonstrated here may become applicable to optimize some of the many FRET biosensors of cellular signaling.