AFM in buffer AFM in air 800nm 800nm Images above are of DNA origami, both on the very same HOPG surface (although very close, not the same 800nmx800nm area). At left is DNA origami on HOPG imaged in liquid, this particular image was taken after HOPG had been exposed to a 0.3 nM origami solution for more than 50 minutes of continual scanning in buffer (1x TAE w/ 12.5mM MgCl2). At right is the very same HOPG surface imaged in air after rinsing and drying. The passivating layer of DNA on the HOPG surface is hardly visible in the liquid scan but serves to protect the DNA origami from disintegration; this layer becomes quite obvious on drying (right).

AFM in buffer AFM in air Images above are of DNA origami, both on the very same HOPG surface (although very close, these are not the same 800nmx800nm area). At left is DNA origami on HOPG imaged in liquid. This particular image was taken after HOPG had been exposed to a 0.3 nM origami solution for more than 50 minutes of continual scanning in buffer (1x TAE w/ 12.5mM MgCl2). At right is the very same HOPG surface imaged in air after rinsing and drying. The passivating layer of DNA on the HOPG surface is hardly visible in the liquid scan but serves to protect the DNA origami from disintegration; this layer becomes quite obvious in the dry scan as a network of dried DNA (right).

1.7um 1.7um 6nm 6nm 0nm 0nm 3 0.7 0.5 2 line profile height/nm dsDNA on mica 2 x dsDNA on mica difference ssDNA vs dsDNA ssDNA on HOPG 1.3 2.7 1.4 0.69 1.5 2.8 0.79 2.4 1.1 0.9 0.83 3.3 1.9 0.75 1.2 0.91 1 1.6 0.97 0.78 3.1 1.7 0.95 2.6 0.96 2.9 0.85 0.81 0.77 0.76 0.71 0.74 0.63 0.98 avg (nm) 1.43 2.82 1.39 sd (nm) 0.12 0.18 0.19 0nm 0nm 3 0.7 0.5 2 line profile height/nm line profile height/nm 0.3 1 0.1 20 40 60 80 100 120 140 10 20 30 40 50 60 70 line profile length/nm line profile length/nm

Images at left indicate heights of dsDNA and ssDNA off of surface of mica (left panel) and HOPG (right panel). Heights were measured as shown in example images with profile plots. 25 measurements were taken such that the averages and standard deviations shown below reflect the magnitude and variability of height of DNA as measured dry by AFM in PeakForce Tapping feedback. The DNA origami shown at left was deposited on mica from a solution that was depleted of surplus staples (diafiltered), exposed to HOPG for 5 minutes (0.3 nM origami solution), then removed and applied to mica. The DNA origami at right was depleted of surplus staples (diafiltered), and applied to HOPG (0.3 nM origami solution) for 5 minutes. Both surfaces were rinsed and blown dry with inert gas. dsDNA on mica 2x deep dsDNA on mica Difference dsDNA 2x deep minus dsDNA ssDNA on HOPG average (nm) 1.43 2.82 1.39 0.85 SD (nm) 0.12 0.18 0.19

U Schematic of cross shaped origami based on Liu (date, ref.). Dashed black box indicates a gap of 2 helices in Left/Right domain that leads to the double high bars at center of origami as seen in AFM (see DNA origami on mica in figure …..). Shown as yellow circles are sites in origami to which biotin is addressed which enables site specific binding of the protein streptavidin (SA). In yellow boxes are biotin modifications attached to 3’ ends of DNA staples (3 of the 4 have tri-ethylene glycol to optimize spacing for SA binding, from Integrated DNA Technologies). 68nm R L D CO-M-9-BIOTIN 5'- GTG CCA AGG AAG ATC GAC ATC CAG ATA GGT T/3BioTEG/ -3' CO-M-16-BIOTIN 5'- TAA GAA AAG ATT GAC CGT AAT GGG CCA GCT T/3BioTEG/ -3' CO-M-74-BIOTIN 5'- AGT AGA AAA GTT TGA GTA ACA /3BioTEG/ -3' CO-M-81-BIOTIN 5'- ATT GAA CCA ATA TAA TCC TGA TTG TCA TTT TG/3Bio/-3’

Schematic of cross shaped origami based on Liu Schematic of cross shaped origami based on Liu. Dashed black box indicates a gap of 2 helices in Lefr/Right domain that leads to the double high bars at center of origami as seen in AFM (see DNA origami on mica in figure …..). Shown in yellow circles are sites in origami to which biotin is addressed which enables site specific binding of the protein streptavidin (SA). In yellow boxes are biotin modifications attached to 3’ ends of DNA staples (3 of the 4 have tri-ethylene glycol to optimize spacing for SA binding) from Integrated DNA Technologies). 68nm CO-M-9-BIOTIN 5'- GTG CCA AGG AAG ATC GAC ATC CAG ATA GGT T/3BioTEG/ -3' CO-M-16-BIOTIN 5'- TAA GAA AAG ATT GAC CGT AAT GGG CCA GCT T/3BioTEG/ -3' CO-M-74-BIOTIN 5'- AGT AGA AAA GTT TGA GTA ACA /3BioTEG/ -3' CO-M-81-BIOTIN 5'- ATT GAA CCA ATA TAA TCC TGA TTG TCA TTT TG/3Bio/-3’