Extended Data Fig. 3: Contribution of the linker to the axial position of single molecules.

a) Sketch showing the systems used to estimate the contribution of the linker. Left: system containing a dsDNA segment with 66 bp, internally labeled at base #45. Right: system containing a dsDNA segment with 45 bp, labeled at one of its end bases. The negative charges are highlighted, since they are responsible for extending outwards the negatively charged dye (ATTO 542), which is attached through a six-atom carbon linker. b) Distribution of angles with respect to the z-axis obtained for the 40–45 bp segment from the MD simulation trajectory of the 51 bp system. The methodology to calculate this angle was analogous to the one described in the caption of Fig. S7d. c) Height distributions for the two systems described in a). d) Representation of the trigonometric calculations performed to retrieve the linker length assuming a model where the linker is stretched, extending outwards of the dsDNA segment (following the direction of the dsDNA segment for the end-labeled case, and oriented perpendicularly for the internally labeled scenario). The 25° angle used was obtained from the histogram shown in b), and the 1.14 nm height difference was extracted from the histograms shown in c).