In this application note, we demonstrate how single-molecule Förster Resonance Energy Transfer (smFRET) can be used to measure nanoscale distances on the EI-FLEX. smFRET is performed on a model DNA duplex labelled with donor and acceptor fluorophores at defined positions, to measure the FRET efficiency and calculate the distance between the two dyes. These data showcase the application of smFRET in probing nanoscale distances on single molecules with Ångström-level precision.
Overview of this application note:
- Three DNA duplex standards that have low, mid and high FRET efficiencies are used to demonstrate the smFRET technique
- Alternating laser excitation is used to identify doubly-labelled DNA complexes and to determine their FRET efficiency and stoichiometry
- FRET efficiencies and known Förster radii are used to calculate nanoscale distances between fluorophore pairs
Figure 1 – Illustration of low-FRET, mid-FRET and high-FRET DNA standards
The donor fluorophore is conjugated on one strand, with an acceptor fluorophore conjugated at various distances that produce low, mid or high-FRET efficiencies
