Agarose gel electrophoresis

Gel characterization of the origami seed or assembled slat filaments was performed using either the ThermoScientific™ Owl™ EasyCast™ B2 or D3-14 electrophoresis system. UltraPure agarose (Life Technologies, 16500500) was melted in 0.5X TBE (45 mM Tris, 45 mM boric acid, 0.78 mM EDTA, 12–16 mM MgCl₂ to match amount in slats experiment) to a concentration of 0.5–2.0 % (w/v). The 0.5–1% gels were typically used to characterize larger assemblies of slats, whereas 2% gels for smaller structures such as the origami seed. The molten agarose was cooled to 65 °C and gel stain was added. All slat assembly reactions were characterized with 10,000× SYBR-Gold (ThermoFisher, S-11494) gel stain added to a final concentration of ~0.183× (i.e., 3 µL per 160 mL molten agarose). Origami seed folding was characterized with 6.25 × 10⁻⁵ % (w/v) ethidium bromide (i.e., 10 µL per 160 mL molten agarose; Bio-Rad, 1610433). Gels were covered with aluminum foil during solidification and running to lessen exposure to ambient light. DNA assemblies were mixed in an excess of agarose gel loading buffer (5 mM Tris, 1 mM EDTA, 30% w/v glycerol, 0.025% w/v xylene cyanol, 12–16 mM MgCl₂; with typically 10 µL loading buffer added to 4–10 µL of each assembly). The mixed samples were loaded onto the gel and separated for 3–4 h at 60 V at room temperature. Control samples for size and densitometry included one or both of the following: first, 0.5–11.2 fmol of folded DNA-origami seed; or second, 0.00625–0.5 μg of Gene Ruler 1 kb Plus DNA Ladder (ThermoScientific™ SM1331). Gel images were captured on a GE Typhoon FLA 9500 fluorescent imager using the SYBR-Gold parameters as given in the Typhoon FLA 9500 Control Software (Version 1.1 Build 1.1.0.187). The photo-multiplier tube (PMT) was set to 300–500 V, with the value varied depending on the amount of sample loaded. Densitometry to quantify relative assembly of DNA bands was performed with ImageJ (v2.0.0-rc-69/1.52i). Background subtraction with a rolling ball radius of 30–60 pixels was performed on linear TIFF images. The GelAnalyzer plugin in ImageJ and wand tool was used to integrate total pixel intensities from lanes of interest. Gel absorbency data collected on different agarose gels were compared to one another by normalizing them with respect to the DNA-origami seed control, as well as the volume of reaction loaded.