Fig. 2: Three-step low-complexity ssDNA Curtains.

a Outline of procedure for preparation of 5′ biotinylated ssDNA substrates with a low-complexity sequence by rolling circle replication of a designed ssDNA template. The template was 5′-/Phos/-TGG GTG TGT GTG TGT GTG TGT GTG GTG GT-3′. The biotinylated primer was 5′-/Biotin/-CAC CCA ACC ACC-3′. b Schematic of procedure for three-step low-complexity ssDNA Curtains. c Schematic of ssDNA Curtains. d Wide-field TIRFM images of ssDNA Curtains at 30-min time point with 0.4 mL/min flow of working buffer containing 10 pM RPA-MeGFP (i), or at 40-min time point with 25-fold RPA-MeGFP (250 pM) with flow on (ii) or off (iii). The green signals from RPA moved back with ssDNA molecules to the barrier when turning off the flow, which confirmed the specific binding of RPA to the ssDNA substrates. e (i) A representative kymograph showing the length dynamics of ssDNA–RPA complex from d. Blue dot line labeled the end tracking positions of a represented ssDNA–RPA complex, and the length analysis of the collective data of many ssDNA molecules was in (ii). N = 45, which was the total trace number of ssDNA molecules end tracking examined over three times DNA Curtains experiments. Data are presented as mean ± SEM in e. The working buffer was 40 mM Tris-HCl (pH 7.5), 2 mM MgCl₂, 0.2 mg/mL BSA, and 150 mM NaCl. Source data are provided as a Source Data file.