Extended Data Fig. 3: Calibration of the optical tweezers assay and control for a nucleation mechanism.
From: Sequence-dependent surface condensation of a pioneer transcription factor on DNA
a, Example confocal image of a dCas9-EGFP (white arrow) labelled λ-DNA molecule held between two 4.42 μm diameter polystyrene beads. b, Mean Intensity profile along the DNA. Gray lines, N = 270 experiments. Red, orange, mean and standard deviation. Blue lines, position of the four dCas9 target sites (Supplementary Table 3 and Supplementary Methods). c, Left, example of an individual dCas9-EGFP molecule. Intensity profile in x and y directions (red circles) fitted to a gaussian function (solid blue line, FWHM = 355.2 and 319.5 nm respectively). Right, examples of 1, 2, 3 and 4 dCas9-EGFP molecules with integrated intensity values of: 156.1, 237.0, 344.2 and 488.8 photon counts. d, e, Probability distribution of integrated intensity values of dCas9 on λ-DNA (N, number of experiments). Red line, fit to a gaussian mixture model (see Methods and Supplementary Table 5). f, Representative force extension curves. Grey lines (N = 30), red line, Worm-like chain model63 (contour length, LC = 16.49 μm (48.514 kbp), persistence length, LP = 50 nm, stretch modulus of K = 1200 pN). g, Histogram of experimental forces. Fexp = 8.22 ± 2.65 pN (mean ± standard deviation). At these forces, the contributions from an intrinsic globular state of the DNA (entropic regime) or the deformation of the double helix structure (enthalpic regime) can be avoided. h–j, Representative confocal images of a mixture of 2.5% Klf4-GFP and 97.5% untagged Klf4 and untagged Klf4 labelled with a small molecule dye (CF488A) at the specified ratios of Klf4:Klf4-dye (Supplementary Methods). [Klf4]=200 nM. k, Background intensity as a function of the loaded concentration. Large symbols, mean, error bars, standard deviaiton. Lines, linear fit (Supplementary Methods and Supplementary Table 4). i, Phase separation assay with 500 nM Klf4-GFP and increasing amounts of Sulforhodamine 101-X (TxRd)-labelled dsDNA oligonucleotides containing 9 Klf4 binding sites. The short oligonucleotides fail to induce Klf4 condensation, indicating that Klf4 condensates do not form on DNA via a nucleation mechanism. Images of the same color have the same contrast settings.
