Monitoring multiple distances within a single molecule using switchable FRET.
The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called 'switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. In contrast, switchable FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules.
Uphoff
Stephan
S
Biological Physics Research Group, Department of Physics, University of Oxford, Oxford, UK.
Holden
Seamus J
SJ
Le Reste
Ludovic
L
Periz
Javier
J
van de Linde
Sebastian
S
Heilemann
Mike
M
Kapanidis
Achillefs N
AN
D019921:Biotinylation; D003198:Computer Simulation; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D009010:Monte Carlo Method; D009690:Nucleic Acid Conformation
D005456:Fluorescent Dyes; D004247:DNA
J Am Chem Soc. 2001 Dec 5;123(48):12123-5
11724636
Biophys J. 2002 May;82(5):2775-83
11964263
Biophys J. 2006 Sep 1;91(5):1941-51
16766620
Proc Natl Acad Sci U S A. 2010 Jan 12;107(2):715-20
20080740
Nat Methods. 2008 Nov;5(11):965-71
18849988
Biophys J. 2008 Jun;94(11):4515-24
18281383
Proc Natl Acad Sci U S A. 2004 Jun 15;101(24):8936-41
15175430
Nat Methods. 2008 Jun;5(6):507-16
18511918
Biophys J. 2010 Nov 3;99(9):3102-11
21044609
Methods Enzymol. 1992;211:353-88
1406315
Nat Biotechnol. 2003 Nov;21(11):1387-95
14595367
Biophys J. 1979 May;26(2):161-93
262414
Biophys J. 2004 Aug;87(2):1328-37
15298935
Phys Rev Lett. 2005 Mar 18;94(10):108101
15783528
Biophys J. 2005 Apr;88(4):2939-53
15653725
Biophys J. 2003 Jun;84(6):3992-4010
12770904
J Fluoresc. 2004 Mar;14(2):145-50
15615040
Proc Natl Acad Sci U S A. 2005 Jun 7;102(23):8186-91
15917329
Biophys J. 2006 Feb 15;90(4):1419-31
16299085
Chemphyschem. 2005 Jan;6(1):74-7
15688649
J Am Chem Soc. 2009 Apr 15;131(14):5018-9
19301868
Angew Chem Int Ed Engl. 2008;47(33):6172-6
18646237
Trends Biochem Sci. 2009 May;34(5):234-43
19362843
Proc Natl Acad Sci U S A. 2009 May 19;106(20):8107-12
19433792
Nat Struct Biol. 2003 Feb;10(2):93-7
12496933
J Mol Biol. 2001 Sep 21;312(3):453-68
11563909
Nat Methods. 2006 Nov;3(11):891-3
17013382
Cell. 2002 Mar 8;108(5):599-614
11893332
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3670-5
10097095
Biochemistry. 2000 May 30;39(21):6317-24
10828944
Curr Opin Struct Biol. 2004 Feb;14(1):10-20
15102444
Methods Mol Biol. 2009;543:503-21
19378183
J Mol Biol. 2006 Mar 17;357(1):173-83
16427082
J Am Chem Soc. 2005 Mar 23;127(11):3801-6
15771514
Nat Methods. 2006 Oct;3(10):793-5
16896339
Angew Chem Int Ed Engl. 2009;48(37):6903-8
19670280
Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18337-42
19020079
Biophys J. 2007 Jan 1;92(1):303-12
17040983
Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2994-8
8464916