To further understand the transcriptome, new tools sensors capable of measuring cellular state, RNA-RNA interactions together with RNA-protein interactions are needed. Although Förster resonance energy transfer (FRET) is an angle- and distance-dependent phenomenon, the majority of FRET measurements have been used to report distances, by assuming rotationally averaged donor-acceptor pairs. Angle-dependent FRET measurements have proven challenging for nucleic acids due to the difficulties in incorporating fluorophores rigidly into local RNA substructures in a biocompatible manner. Genetically encodable fluorescence turn-on RNA aptamer tags can orient their cognate fluorophores with respect to local RNA structures, and report angular-resolved FRET. The application of fluorescence turn-on aptamers to cellular FRET opens the way to improved structural interpretation of ensemble and single-molecule FRET measurements of RNA in living systems.
The use of RNA Mango in angular reolved FRET is demonstrated here