Buffers and gels

TAE BUFFER

Materials	Concentration
Tris-acetate (pH8.3)	40mM
EDTA	1mM

TAE-MG BUFFER

Materials	Concentration
Tris-acetate (pH8.3)	40mM
EDTA	1mM
Mg(OAc)2	10mM

10％PAGE

Materials	Quantity
water	4.8ml
40×TAE-Mg	188ul
30% m/v acrylamide	2.5ml
TEMED	7.5ul
10% APS	75ul

15％PAGE

Materials	Quantity
water	3.55ml
40×TAE-Mg	188ul
30% m/v acrylamide	3.75ml
TEMED	7.5ul
10% APS	75ul

Electrophoresis

Samples were loaded on the PAGE gels described above (10% for TPS [three point star] unit, 15% for joints). TAE was used as running buffer. After 15V for 15 hour electrophoresis, PAGE gels were stained by Ethidium bromide. Stained DNA was observed by UV illuminator (365 nm).

The electrophoresis was conducted as below.

	T1	T2	T3	T1+T2	T2+T3	T3+T1	T1+T2+T3
T1(1uM)	+			+		+	+
T2(3uM)		+		+	+		+
T3(3uM)			+		+	+	+

	J1	J2	J1+J2
J1(2uM)	+		+
J2(2uM)		+	+

These DNA strands were hybridized by 90 ºC for 5 min, 90 ºC for 5 min, 65 ºC for 5 min, 50 ºC for 5 min, 37 ºC for 5 min,, and 25 ºC for 5 min. The DNA after annealing was stored at 4 ºC.

Formation of DNA tile sheets

Under the following conditions DNA was occluded and then annealed to create the DNA tiles.

DNA concentrations are as follows.

	TPS+dsDNA joint	TPS+dsDNA joint
T1	1uM	1uM
T2	3uM	3uM
T3	3uM	3uM
J1	1.5uM	1.5uM
J2	1.5uM	none

For annealing, we conducted a temperature change program as the following protocol using ThermoGradient96 (BIOMETRA).

DNA tile sheets observation by Fluorescence microscopy

1/1000 dense MIDORI GREEN (Nippon Genetics) was mixed with newly-formed 5 uL of DNA tile before observation. DNA tiles were observed using a fluorescent microscope Axiovert 200M (Carl Zeiss, Jena, Germany) with a CMOS camera ORCA-Flash4.0 V2 (Hamamatsu Photonics, Shizuoka, Japan). A fluorescent filter for GFP (Zeiss) was used to detect DNA stained with MIDORI GREEN.

DNA tile wrapping

First, newly-formed 5 uL of DNA was mixed with 0.5uL of 10 ug/mL Ethidium bromide (EtBr, Nacalai Tesque). Fluorescent images were captured using the same set-up as above. A fluorescent filter for GFP (Zeiss) was used for the 200nm beads, and a Cy3.5 filter (ThorLab) was used for EtBr stained DNA.
Fluorescent beads were mixed with the DNA-tile stained with EtBr. The beads were FluoSpheres (carboxylate modified. 0.2 um in diameter, 2 mM surface azide group; Ex/Em= 505/515 nm, Invitrogen). Then, 20 uM at final of Capture DNA was added to wrap the object.

Atomic Force Microscopy (AFM)

The DNA tile solution was spotted in 1uL on mica and the nano-sized structures were observed following the AFM manual for Veeco Manifold Multimode V).