This application note was produced in collaboration with the Hill Lab at the University of York.
In this application note, we demonstrate how single-molecule Förster Resonance Energy Transfer (smFRET) can be performed using the EI-FLEX to resolve a previously unknown RNA virus riboswitch mechanism. Here, Betts et al. performed smFRET on the EI-FLEX system, alongside X-ray crystallography and small-angle X-ray scattering (SAXS), uncovering protein-dependent RNA pseudoknot formation that acts as a stimulatory element, regulating programmed –1 ribosomal frameshifting (PRF) in Theiler’s murine encephalitis virus (TMEV).
Overview of this application note:
- smFRET adds complementary information to structural techniques such as X-ray crystallography and SAXS to elucidate RNA conformational changes upon protein binding
- Burst variance analysis reveals that pseudoknot formation is a binary event dependent on protein 2A binding, rather than continuous conformational shifts
- smFRET provided insights alongside binding analysis that elucidated which RNA bases are crucial for protein 2A binding
Figure 1 – Different dye placements for smFRET act as end or internal reporters
Schematics of protein labelling for smFRET, demonstrating how FRET efficiencies are influenced by dye placement and protein-induced conformational changes.Â
