Introduction 1 E 1 \documentclass[12pt]{minimal}
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\begin{document}$$E = \frac{1}{{1 + {\left( {R \mathord{\left/ {\vphantom {R {R_{0} }}} \right. \kern-\nulldelimiterspace} {R_{0} }} \right)}^{6} }}$$\end{document} R R 0 2 4 5 in vivo 6 7 8 9 10 Although the conceptual beauty of FRET studies is undisputed, there are a number of important caveats in single-molecule FRET studies of biomolecules, such as fluorophore blinking, photobleaching and sample immobilization. Here, we addressed these issues. spFRET microscopy on mononucleosomes revealed two dominant types of dynamics: acceptor blinking and intramolecular rearrangements that we attribute to DNA breathing, which only became apparent after suppression of blinking. Upon immobilization, we observed three different populations: 90% of the nucleosomes dissociated or represented donor-only species, and 10% remained intact. Of these fully wrapped nucleosomes, 97% showed stable FRET on timescales between 0.01–10 s, while 3% showed dynamics with a dwell time of 120 ms that we attribute to conformational changes in the nucleosome. Material and methods DNA preparation 11 T C 1 12 E R 0 R 0 Fig. 1 a b c  Nucleosome reconstitution Recombinant histone octamers were mixed with the DNA construct at a 1:1 ratio, in TE (1 mM EDTA, 10 mM TRIS pH 8.0) and 2 M NaCl. Mononucleosomes were reconstituted by salt dialysis against 0.85, 0.65, 0.5 and finally against 0.1 M NaCl, all buffered with TE. Bulk fluorescence measurements (ratio) A 13 2 \documentclass[12pt]{minimal}
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\begin{document}$$E = {\left( {\frac{{\varepsilon ^{A}_{{615}} }}{{\varepsilon ^{D}_{{515}} d^{ + } }}\frac{{F^{A}_{{515}} }}{{F^{A}_{{615}} }} - \frac{{\varepsilon ^{A}_{{515}} }}{{\varepsilon ^{D}_{{515}} d^{ + } }}} \right)}$$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$$ \varepsilon ^{A}_{\lambda }  $$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$$ \varepsilon ^{D}_{\lambda }  $$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$$ F^{A}_{\lambda }  $$\end{document} + + Single-molecule FRET measurements d n + 2 2 14 2 2 15 Data analysis 2 3 \documentclass[12pt]{minimal}
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\begin{document}$$E = \frac{{I_{A} }}{{I_{A}  + \gamma I_{D} }}$$\end{document} I A I D \documentclass[12pt]{minimal}
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\begin{document}$$ \gamma  = \frac{{\phi _{A} \eta _{A} }} {{\phi _{D} \eta _{D} }} $$\end{document} Φ A Φ D η A η D γ γ 4 \documentclass[12pt]{minimal}
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\begin{document}$$E = 1 - {I_{D} } \mathord{\left/ {\vphantom {{I_{D} } {I_{{D0}} }}} \right. \kern-\nulldelimiterspace} {I_{{D0}} }$$\end{document} I D0 3 4 5 \documentclass[12pt]{minimal}
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\begin{document}$$\gamma  = \frac{{I_{A} }}{{I_{{D0}}  - I_{D} }}$$\end{document} Experimental results Bulk fluorescence spectra reveal proper reconstitution of mononucleosomes 2 2 16 Fig. 2 a b  2 1 spFRET microscopy reveals individual nucleosomes together with a large population of dissociated nucleosomes 3 3 Fig. 3 a arrows b  3 17 18 19 20 21 On the 10% immobilized mononucleosomes showing FRET, irreversible loss of FRET was only found after photobleaching, implying that their nucleosomal structure remained intact after immobilization. Single-molecule fluorescence footprint of individual nucleosomes 4 3 3 2 5 G η D η A 4 Fig. 4 top panels a b middle panel bottom panels c d  4 Acceptor blinking is the dominant source of spFRET dynamics 4 E E 10 7 22 3 σ tot 23 6 \documentclass[12pt]{minimal}
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\begin{document}$$\sigma _{{{\text{tot}}}}  = {\sqrt {G^{2} F^{2} S\Phi  + G^{2} F^{2} D + \sigma _{{\text{R}}} } }$$\end{document} G F S Φ D σ R σ F \documentclass[12pt]{minimal}
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\begin{document}$$F_{{\alpha ,\nu _{1} ,\nu _{{12}} }}  \geqslant \frac{{\sigma ^{2} }}{{\sigma ^{2}_{{{\text{tot}}}} }},$$\end{document} α ν 1 ν 2 σ σ tot Suppression of blinking 24 5 Fig. 5 a b top bottom c d top bottom  25 5 5 A fraction of the immobilized nucleosomes shows dynamics clearly distinct from blinking 6 6 6 6 Fig. 6 a b grey bars c d top panels bottom panels insets e f  Discussion and conclusion 10 Our single-molecule measurements revealed at least three subpopulations in the reconstituted and immobilized nucleosome sample: 90% of the fluorophores represented dissociated nucleosomes or donor only species, 10% represented intact nucleosomes. Of these, 97% remained stable on time-scales ranging from 10 ms to 10 s of seconds, while 3% showed intervals with reduced FRET efficiency and a lifetime of 120 ms clearly distinct from blinking. 26 21 27 10 8 9 −1 8 9 Our findings demonstrate that experimental conditions can have a profound impact on the data obtained when probing nucleosome structure and conformational dynamics. Immobilization effects and blinking dynamics have to be accounted for, and where possible suppressed in order to extract biologically relevant data from spFRET experiments. We have shown that DNA breathing kinetics obtained from carefully optimized spFRET experiments approaches values obtained from bulk experiments, opening the way to more complex single-molecule studies of chromatin dynamics.