Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule's signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.
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We thank H. Yardimci and J.J. Loparo for assistance with experiments and critical reading of the manuscript and C.G. Havens (Harvard University) for sharing advice and reagents. This work was supported by grants from the US National Institutes of Health (NIH) (GM077248), American Cancer Society (RSG0823401GMC) and Netherlands Organization for Scientific Research (NWO; Vici 680-47-607) to A.M.v.O. as well as by grants from the NIH (GM62267) and American Cancer Society (RSG0823401GMC) to J.C.W. A.B.L. was supported by the NIH and National Institute of General Medical Sciences Molecular Biophysics Training Grant (T32 GM008313).
The authors declare no competing financial interests.
Supplementary Text and Figures
Supplementary Figures 1–8, Supplementary Tables 1–3 and Supplementary Methods (PDF 1007 kb)
PhADE imaging of Fen1KikR under conditions when only one origin fires reveals the growth rate of individual replication bubbles.
D179A Fen1KikR was imaged (as in Fig. 2b) using PhADE between 5 and 25 min after NPE addition (time stamps). During this time, D179A Fen1KikR signals appeared, grew in length, and some eventually split as forks traveled far apart. Some D179A Fen1KikR signals were lost as DNA tethers broke and DNA compacted. The extracts were washed out 25 min after NPE addition, dig-dUTP incorporation was detected and DNA was stained with an intercalating dye. (AVI 5321 kb)
PhADE imaging of D179A Fen1KikR reveals the timing and distribution of initiation events.
D179A Fen1KikR was imaged using PhADE (as in Fig. 3a) between 2.5 and 10 min after NPE addition (time stamps). During this time, D179A Fen1KikR signals appeared, grew and merged, which we interpret as replication initiation, elongation and termination. Some molecules were lost as DNA tethers broke and DNA compacted. A kymograph of the molecule labeled #1 in the first frame of the movie is shown in Figure 3a. All molecules highlighted with cyan boxes in the first frame were analyzed for Fig. 3b–d. Arrows indicate QDot 605-Biotin that are used as fiduciary markers. (AVI 1942 kb)
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Loveland, A., Habuchi, S., Walter, J. et al. A general approach to break the concentration barrier in single-molecule imaging. Nat Methods 9, 987–992 (2012). https://doi.org/10.1038/nmeth.2174
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