Journal
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS
Volume 38, Issue 4, Pages 395-405Publisher
SPRINGER
DOI: 10.1007/s00249-008-0383-z
Keywords
Fluorescence; FRET; Single molecule; Flexibility; DNA/RNA duplex
Categories
Funding
- BBRSC
- Wellcome Trust VIP grant
- Biotechnology and Biological Sciences Research Council [BBS/B/0367X] Funding Source: researchfish
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Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Forster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07-0.2 (on a scale of 0-1). Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.
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