This application note was produced in collaboration with the Antony lab at St Louis University School of Medicine.
In this application note, we demonstrate how single-molecule Förster Resonance Energy Transfer (smFRET) can be performed using the EI-FLEX to measure the end-to-end distances of single-stranded DNA (ssDNA) molecules. Here, Chadda et al. explore ssDNA molecules of varying lengths, investigating the impact of binding to replication protein A (RPA)1. Existing models using crystal structures or bulk methods have failed to reach consensus on whether binding causes DNA wrapping or stretching, demonstrating that in-solution and single-molecule methods are crucial for elucidating dynamic conformations and nanoscale distances.
Overview of this application note:
- End-to-end distances of a variety of ssDNA molecules were calculated from FRET efficiency values, showing linear relationships between number of bases and distance
- Data captured on the EI-FLEX was in good agreement with distances calculated using the Picoquant MT-200 instrument
- A reduction of end-to-end distance of 2.6 Å was observed upon addition of RPA, which was maintained irrespective of ssDNA length, indicating that DNA wraps around RPA, rather than being stretched
Figure 1 – FRET efficiency and end-to-end distance show linear relationship to ssDNA length
A) FRET efficiency plotted against number of bases in ssDNA molecules
B) End-to-end distance (nm) plotted against number of bases in ssDNA molecules
Figure taken from Chadda et al.1
