Supplementary Information

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1 Supplementary Information STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA Iddo Heller, Gerrit Sitters, Onno D. Broekmans, Géraldine Farge, Carolin Menges, Wolfgang Wende, Stefan W. Hell, Erwin J. G. Peterman, Gijs J. L. Wuite Supplementary Information Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Note 1 Supplementary Note 2 Title Single fluorophore bleaching traces Contribution of DNA fluctuations to spatial resolution Effect of ROXS on DNA mechanics STED nanoscopy of DNA that is densely coated with TFAM Number of photons per line scan as function of STED power Fit of experimental localization precision No evidence for enhanced photonicking by STED Retarded TFAM diffusion

2 Supplementary Figure 1: Single fluorophore bleaching traces Supplementary Figure 1 Single fluorophore bleaching traces. (a-c) Time traces of bleaching steps of respectively EcoRV-Atto647N, Sytox orange, and an EGFP-labeled protein, measured by confocal microscopy on optically stretched DNA.

3 Supplementary Figure 2: Contribution of DNA fluctuations to spatial resolution Supplementary Figure 2 Resolution scaling with STED power. The solid line corresponds to the fitted of Figure 3g, where we used as fit parameter and. By suppressing DNA fluctuations, the spatial resolution can be improved towards the dashed line, which uses, for instance by applying high tension. The difference between the two curves highlights the apparent reduction of the spatial resolution due to the fluctuations of the DNA, under the experimental conditions of Figure 3g (at 4 pn tension).

4 Supplementary Figure 3: Effect of ROXS on DNA mechanics Supplementary Figure 3 Effect of ROXS and enzymatic oxygen removal (GLOX) on DNA mechanics. The black curve shows a force-distance curve of λ DNA in a 20 mm tris buffer with 100 mm NaCl. The magenta curve shows a force-distance curves of λ DNA in the same buffer supplemented with 1 mm methylviologen and 2 mm trolox (ROXS), with enzymatic oxygen removal by glucose oxidase, catalase, and glucose (GLOX). At forces over 30 pn, a small length increase (<5%) is apparent in presence of ROXS GLOX. Although this could be suggestive of intercalation of the ROXS components, it is a strikingly weak effect compared to intercalation by, for example, ethidium, YoYo-1, and ruthenium complexes, 33,34 which induce far more drastic changes to force-distance curves at concentrations that are 5 to 6 orders of magnitude smaller than those used here. Furthermore, the overstretching plateau, which is known to be sensitive to intercalating and groove binding buffer constituents, occurs at ~65 pn for all curves.

5 Supplementary Figure 4: STED nanoscopy of DNA that is densely coated with TFAM Supplementary Figure 4 STED nanoscopy of DNA that is densely coated with TFAM. (a) Confocal microscopy image of TFAM-Atto647N filaments at high density on DNA. (b) STED kymograph of the section of the DNA imaged in (b). STED power 16 mw. (c) Cumulative intensity profile as indicated in a (blue) and b (red), comparing confocal and STED profiles. The STED intensity profile exhibits peaks at ~100 nm spacing, indicating that STED can indeed be used to image dense DNA-bound protein filaments with unprecedented resolution.

6 Supplementary Figure 5: Number of photons per line scan as function of STED power Supplementary Figure 5 Number of photons per line scan of a single DNA-bound protein as function of STED power. The symbols show the experimentally determined number of photons per line scan as function of STED power corresponding to the data of Figure 4d. The line is the fit of the model described in the Online Methods, which takes into account the dependence of the number of photons on the width of the intensity distribution and the loss of photons due to a non-zero intensity in the line-shaped minimum of the STED beam.

7 Supplementary Figure 6: Fit of experimental localization precision Supplementary Figure 6 Fit of the experimental localization precision of Figure 4d. The symbols correspond to the measured localization precision of Figure 4d. The line is a fit of the additional noise term, using the model described in the Online Methods, yielding = 3.6 nm.

8 Supplementary Note 1: No evidence for enhanced photonicking by STED An intrinsic advantage of performing STED imaging in optical tweezers is that the structural integrity of the DNA and the presence of nicks can be directly evaluated by monitoring the force on the microspheres and (over)stretching the DNA: A double-stranded break in the DNA would break the link between the two beads, leading to zero force on the beads, and loss of the DNA in the image. No such double-stranded breaks have been observed during confocal or STED imaging. Photonicking (single-stranded break), however, would keep the link between the beads intact. The presence of nicks can be evaluated by overstretching DNA in medium to low ionic conditions: As the double-helix melts during overstretching, the presence of a nick would lead to a loss in the link between the two beads. 35 Indeed, no significant additional photo nicking was observed after confocal or STED imaging of labeled proteins on DNA. Although there is a finite chance that single nicks in long molecules may not be observed in an incomplete overstretching experiment, the lack of observation of additional nicks implies that the rate of photonicking is not elevated to the extent that numerous nicks exist after STED imaging.

9 Supplementary Note 2: Retarded TFAM diffusion The diffusion constant of monomeric TFAM, 29 D ref 0.08 µm 2 /s is slower than diffusion of typical proteins that undergo rotation-coupled translation along the DNA helix (D µm 2 /s). 36 Assuming large values of the TFAM radius R = 3 nm and the width of its helical path R oc = 2 nm, and a typical average free energy barrier for sliding ε = k B T, we obtain a lower estimate of the TFAM diffusion constant in case of rotation-coupled translation of D = 0.5 µm 2 /s, which is indeed significantly higher than D ref. 37 This suggests that TFAM translocation is heavily retarded by a strong DNA-protein interaction. This strong interaction can be related to the elongated structure of TFAM with three DNA-interaction sites, and is consistent with its ability to bend and compact mitochondrial DNA. 27,28 To a first, overly simplified, approximation, D is considered to be inversely proportional to the number of TFAM monomers in an oligomer, N TFAM, such that. The solid grey line in Figure 6d represents the expected distribution of D if we assume a mix of equal populations of oligomers with N TFAM = 1,2,3,,12, which is in reasonable agreement with the measured diffusion data. Direct analysis of the correlation of D with I norm (not possible here due to the low labeling ratio) could further elucidate the scaling of D with N TFAM at high protein density.

10 SUPPLEMENTARY REFERENCES 33. Vladescu, I.D., McCauley, M.J., Nun, M.E., Rouzina, I. & Williams, M.C. Quantifying forcedependent and zero-force DNA intercalation by single-molecule stretching. Nat. Methods 4, (2007). 34. Murade, C.U., Subramaniam, V., Otto, C. & Bennink, M.L. Interaction of oxazole yellow dyes with DNA studied with hybrid optical tweezers and fluorescence microscopy. Biophys. J (2009). 35. van Mameren, J. et al. Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging. Proc. Natl. Acad. Sci. USA 106, (2009). 36. Blainey, P.C. et al. Nonspecifically bound proteins spin while diffusing along DNA. Nat. Struct. Mol. Biol. 16, (2009). 37. Bagchi, B., Blainey, P.C. & Xie, X.S. Diffusion constant of a nonspecifically bound protein undergoing curvilinear motion along DNA J. Phys. Chem. B 112, (2008).