Nb bbvci

Closed circular (cc) DNA beads were prepared as described (Higashi et al, 2012 (link)). Briefly, single-stranded DNA templates prepared using pBluescript II KS(−) were annealed with a biotinated oligonucleotide primer (5′-CGCCTTGATCGT [biotin-dT]GGGAACCGGAGCTGAATGAAGC-3′). After second-strand synthesis and ligation, covalently cc double-stranded DNA was purified by CsCl/EtBr density gradient centrifugation, incubated with streptavidin, and bound to biotin-sepharose beads to produce the cc DNA beads (around 100 ng per 1 μl bed volume of beads). A single nick was introduced by Nb.BbvCI (New England Biolabs) treatment to produce the ncDNA beads. As control beads, biotin-sepharose beads were incubated with streptavidin alone (sa-beads).

Thrombin, insulin, and other reagents, unless specified, were purchased from Sigma Aldrich (St. Louis, MO). Iridium chloride hydrate (IrCl₃.xH₂O) was purchased from Precious Metals Online (Australia). Exonucease I (ExoI), Exonucease III (ExoIII) and Nb.BbvCI were purchased from New England Biolabs Inc. (Beverly, MA, USA). All oligonucleotides were synthesized by Techdragon Inc. (Hong Kong, China). The protein HIF-1α is purchased from Sino Biological Inc. (Beijing, China). The DNA secondary structures are simulated by software DNAMAN 6.0.3.99 (Lynnon Biosoft, America).

ATP, dithiothreitol (DTT), DNase I, ethidium bromide, glyoxal and RNase A were purchased from Sigma Aldrich (St Louis, MO). Acrylamide, ampicillin, chloroform, isopropyl beta-D-thiogalactoside, sodium chloride and sodium citrate were purchased from Fisher Scientific (Pittsburgh, PA). BbvCI, EcoRV, Nb.BbvCI, NdeI, Nuclease Bal-31, T4 DNA Ligase, low molecular weight DNA ladder and 100 bp DNA ladder were purchased from New England Biolabs (Ipswich, MA). Proteinase K was purchased from Roche Molecular Biochemicals (Mannheim, Germany). All other chemicals were purchased from VWR International (West Chester, PA).

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.

MseI, Nb.BbvCI, Proteinase K, T4 DNA Ligase, low molecular weight DNA ladder, and 100 bp DNA ladder were purchased from New England Biolabs (Ipswich, MA, USA). Adenosine triphosphate (ATP), antifoam 204, dithiothreitol (DTT), ethidium bromide, and RNase A were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acrylamide, ampicillin, chloroform, and sodium chloride were purchased from Fisher Scientific (Pittsburgh, PA, USA). All other chemicals were purchased from VWR International (West Chester, PA, USA).