Monitoring multiple distances within a single molecule using switchable FRETRETRET英文文献.pdfVIP

Articles monitoring multiple distances within a single molecule using switchable Fret 1 1 1 1 2 Stephan Uphoff , Seamus J Holden , Ludovic Le Reste , Javier Periz , Sebastian van de Linde , Mike Heilemann2 Achillefs N Kapanidis1 the analysis of structure and dynamics of biomolecules is have been developed, including three-color FRET8,9 and three- important for understanding their function. toward this color ALEX10 techniques. Such experiments are complicated: they . d aim, we introduce a method called ‘switchable Fret’, which require site-specific labeling with fluorophores exhibiting large e v combines single-molecule fluorescence resonance energy spectral overlap for FRET yet having sufficient spectral separation r e transfer (Fret) with reversible photoswitching of fluorophores. to allow detection in three or more separate emission regions. s e r typically, single-molecule Fret is measured within a single Moreover, FRET between multiple fluorophores follows a cascade s t donor-acceptor pair and reports on only one distance. Although of energy-transfer processes, complicating data analysis and inter- h g multipair Fret approaches that monitor multiple distances pretation9. As a result, multicolor FRET methods cannot be easily i r l have been developed, they are technically challenging and extended, and as such, they have not realized their potential. l A . difficult to extend, mainly because of their reliance on Here we describe