title: Single-molecule three-color FRET with both negligible spectral overlap and long observation time.
abstract: Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy.
authors.affiliation: Department of Physics and Astronomy, Seoul National University, Seoul, Korea.
authors.forename: Sanghwa
authors.initials: S
authors.lastname: Lee
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Lee
authors.forename: Sungchul
authors.initials: S
authors.lastname: Hohng
mesh_terms: D000042:Absorption; D001483:Base Sequence; D003116:Color; D004247:DNA / Q000737:chemistry / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007834:Lasers; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010100:Oxygen / Q000737:chemistry; D038761:Photobleaching; D013997:Time Factors
chemical_list: D004247:DNA; D010100:Oxygen
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11176-81
references.pmid: 18676615
references.citation: Biophys J. 2004 Aug;87(2):1328-37
references.pmid: 15298935
references.citation: Cell. 1988 Oct 7;55(1):79-89
references.pmid: 3167979
references.citation: Nat Struct Biol. 1999 Oct;6(10):913-7
references.pmid: 10504723
references.citation: Nature. 1989 Oct 26;341(6244):763-6
references.pmid: 2797209
references.citation: Methods. 2001 Sep;25(1):78-86
references.pmid: 11558999
references.citation: Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6264-8
references.pmid: 8692803
references.citation: Nat Methods. 2008 Jun;5(6):507-16
references.pmid: 18511918
references.citation: Methods Enzymol. 1992;211:353-88
references.pmid: 1406315
references.citation: Biophys J. 2008 Mar 1;94(5):1826-35
references.pmid: 17921203
references.citation: Biophys J. 2006 Feb 15;90(4):1419-31
references.pmid: 16299085
references.citation: Chemphyschem. 2005 Jan;6(1):74-7
references.pmid: 15688649
references.citation: Nat Struct Biol. 2003 Feb;10(2):93-7
references.pmid: 12496933
references.citation: Nat Methods. 2006 Nov;3(11):891-3
references.pmid: 17013382
references.citation: J Phys Chem B. 2007 Jan 18;111(2):321-6
references.pmid: 17214479
references.citation: Angew Chem Int Ed Engl. 2007;46(12):1999-2001
references.pmid: 17279589
references.citation: J Mol Biol. 2004 Aug 13;341(3):739-51
references.pmid: 15288783
references.citation: Proc Natl Acad Sci U S A. 2004 Jun 15;101(24):8936-41
references.pmid: 15175430
references.citation: Biophys J. 2007 Jan 1;92(1):303-12
references.pmid: 17040983
references.citation: Science. 2007 Oct 12;318(5848):279-83
references.pmid: 17932299
references.citation: Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):4985-90
references.pmid: 20194794
references.citation: Nano Lett. 2005 Jul;5(7):1509-14
references.pmid: 16178266