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Analytical Chemistry

Going The Distance

Structural Biology: Switchable fluorescence technique measures multiple distances in single molecules

by Celia Henry Arnaud
September 8, 2010 | A version of this story appeared in Volume 88, Issue 37

A new version of fluorescence resonance energy transfer (FRET) makes it possible to monitor multiple distances within individual molecules more easily than ever before (Nat. Meth., DOI: 10.1038/nmeth.1502).

Conventional FRET provides distance information because an acceptor chromophore fluoresces only when it is close enough to be excited by a nearby donor. Although FRET can be carried out with multiple acceptors, such experiments are difficult. By developing switchable FRET, Achillefs N. Kapanidis of the University of Oxford and coworkers there and at Bielefeld University, in Germany, avoid these complications by using acceptors that can be turned on and off with light. They can then install multiple copies of identical acceptors on individual biomolecules.

Using the cyanine dyes Cy3B and Cy5 as donor and acceptor, respectively, they monitor the structure of a DNA-protein complex and probe the conformational dynamics of a DNA Holliday junction—a structure formed from four strands of DNA—from two perspectives.

"This simplified switching method represents a significant and neat addition to the single-molecule toolbox and will nicely complement labeling and other advances in the multicolor single-molecule FRET field," says Ashok Deniz of Scripps Research Institute, one of the developers of three-color FRET.


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