Generating DNA Unwinding/Translocation Tracks

To generate unwinding/translocation tracks of different lengths^{18 (link)}, 600 and 4000 bp tracks were obtained using standard PCR reactions (Supplementary Table 11, IDT) and nicked using Nt.BbvCI for the Biotin-terminated track and Nb.BbvCI for the Digoxigenin-terminated one (enzymes from New England Biolabs), resulting in complementary 29-nucleotides flanked with three nucleotides (5′-TGC-3′). For the symmetric geometry, the 600 biotin and digoxigenin tracks were mixed at equal molar ratios for DNA annealing, creating a ∼1200 bp fragment. For the asymmetric geometries, 4000 bp tracks were annealed to complementary purchased oligonucleotides with the opposite modification (Supplementary Table 11, HPLC purified, IDT). This resulted in asymmetric tracks with 4000 bps and ∼35 nt single-stranded DNA on opposite sides. A ∼250 dsDNA stem containing the “601” sequence^{48 (link)} was amplified from a plasmid (a generous gift from Daniela Rhodes, MRC, Cambridge, UK) using primers listed in Supplementary Table 11, digested using DraIII-HF (New England Biolabs) overnight according to the manufacturer’s instructions, and ligated to the tracks.