Journal
ANALYTICAL CHEMISTRY
Volume 80, Issue 22, Pages 8389-8397Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac8009092
Keywords
-
Categories
Funding
- European Union
- Biotechnology and Biological Research Council of the UK
Ask authors/readers for more resources
Single-molecule fluorescence resonance energy transfer (FRET) is commonly used to probe different conformations and conformational dynamics of single biomolecules. However, the analysis of raw burst traces is not always straightforward. The presence of a zero peak and the skewness of peaks at high and low FRET efficiencies in proximity ratio histograms make the accurate evaluation of the histogram a challenging task. This is further compounded by the difficulty associated with siting two fluorophores in optimal range of each other. Here we present an alternative method of analysis, based on handling coincident FRET photon bursts, that addresses these problems. In addition, we demonstrate methods to enhance coincidence levels and thus the accuracy of FRET determination: the use of dual-color excitation, including direct excitation of the acceptor fluorophore; the addition of a remote dye to the biomolecule, not involved in the FRET process; or a combination of the two. We show the advantages of dual excitation by studying several labeled double-stranded DNA samples as FRET models. Ibis method extends the application of single-molecule FRET to more complicated biological systems where only a small fraction of complexes are fully assembled.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available