Meller Group : Single Molecule Biophysics & Nano-biotechnologyTechnion Logo

Double-stranded DNA as a "spectroscopic ruler" for single molecule FRET

The ability to probe biomolecular dynamics at the level of an individual complex has allowed scientists to explore biological processes with unprecedented detail. In contrast to bulk measurements, single-molecule experiments provide an opportunity to observe the detailed kinetics of individual biomolecules that might otherwise remain masked by ensemble averages. In particular, single-molecule Fluorescnce resonance energy transfer (sm-FRET) has become a popular technique due to its relative simplicity, high sensitivity, and ability to follow the dynamics of individual complexes over timescales relevant for many biomolecular processes.

The quantitative evaluation of the FRET efficiency must take into account a number of factors, including the possibility of dye-dye interactions (other than that of FRET) at short distances. We use dsDNA molecules as a model system to position donor-acceptor pairs at well-defined distances (see Figure 1), and measured the FRET efficiencies. An extensive study of the dependence of FRET efficiency on inter-dye distances from ensemble and immobilized single-molecule measurements reveals outstanding agreement between these two and the model for donor-acceptor separations larger than ~5 nm. At shorter distances, however, a systematic inconsistency between the bulk FRET and the model prediction prompted us to closely examine the single-molecule traces. We find that at these distances donor-acceptor pairs exhibit correlated intensity fluctuations, which can be observed by either donor excitation through FRET, or by direct acceptor excitation. Two single molecule traces are shown in Figure 2, at long distances (D12A) and shorter distances (D6A). The donor and acceptor intensities are shown in green and red. respectively, and the FRET efficienies in blue.

Figure 3, displays the FRET efficiency measured in the single molecule experiment as well as in bulk. The FRET prediction based on the dsDNA model is shown as a solid black line. We observe excellent agreement with the single-molecule data down to distances of ~3 nm.

References:

  1. Di Fiori N. and A. Meller.(2010) The effect of dye-dye interactions on the spatial resolution of single-molecule FRET measurements in nucleic acids. Biophys J. 98(10), 2265-72.

Figure 1

 

FRET trace dsDNA
Figure 2

E vs R

Figure 3