Material & Method

Protocol

Fig. 1 Outlines of our experiments

Fig. 1 shows a brief outline of our experiments.

Origami Design

We designed four types of origami by using caDNAno. Their schematics are summarized in Fig. 2. In addition, source codes of the designs in json format are available in the lists below.

(+/+).json (+/-).json (-/+).json (-/-).json

Fig. 2 caDNAno designs

By using different sets of Shaft-staples, it is possible to change the angles of the shaft. This time, we prepared four sets of staple strands for the shaft. here.

Sample Preparation

1.Mixing

Our DNA origami samples were prepared by mixing a stoichiometric quantity as shown in Table 1.

Table 1 Regular sample composition

	Final Concentration
Shaft Staples	100nM
Cylinder Staples	20nM
M13mp18	2nM
Buffer	1×TAE/SEA

SEA buffer contains a mixture of irons, Na⁺, Mg²⁺, Ca²⁺

To find out the most suitable condition, we changed the proportion of concentration.(Table 2)

Table 2 Another sample composition

	Final Concentration
Shaft Staples	100nM
Cylinder-Staples	50nM
M13mp18	2nM
Buffer	1×TAE/Mg²⁺10mM

2 Annealing

We annealed DNA origami samples by the following conditions (Fig. 3). Furthermore, we tried different annealing conditions as shown in Fig.4-5 to figure out the best annealing condition

Electrophoresis

Gel electrophoresis was conducted by a standard procedure as follows.

Mix 1×TAE buffer 100mL and agarose gel powder (Takara Japan)0.80g.
Heat the mixture by microwave for about 30 seconds.
Pour the heated mixture into the plastic case and leave it for about 30 minutes.
Set the gel on electrophoresis chamber (Mupid-2plus, Mupid, JAPAN).
Mix 5µL of sample and 1µL of 6×Loding buffer.
Apply 6µL of each sample to wells.
Apply 50V for about 80 minutes.
Stain the gel by SYBR GOLD for about 20 minutes.
Observe fluorescence by gel imager (ChemiDoc MP, BIO-RAD, USA).

AFM Imaging

AFM imaging was conducted as follows. We used an observation buffer in Table 3.

Fix mica (Furuuchi Chemical Corporation, JAPAN) on a glass plate
Put 2µL of sample solution on mica
Wait for over 10 minutes
Set up AFM (Nano Live Vision, RIBM, JAPAN)
Observe samples with tapping mode (Micro Cantilever, OLYMPUS, JAPAN)

Table 3 Observation buffer composition

	Concentration[mM]
Tris/Tris-HCl	20
Mg²⁺	12.5

The histogram in Experiment page was based on AFM images below. (Fig. 6)

Fig. 6(a)AFM images of (-/-), 2240 ×1680nm
(b)AFM images of (+/+), 2240 ×1680nm
(c) AFM images of (+/-), 2240 ×1680nm

Cryo-TEM

All cryo-TEM images were taken by Terabase Inc. at Okazaki, Aichi, Japan (http://www.terabase.co.jp/English.html). Fig. 10 in Experiment page was trimmed from Fig. 7 which is shown below.

Fig. 7 TEM observation (1333.68×1333.68, magnification: ×30k.)

Reconstructed cryo-TEM images in cross and longitudinal section were created from 16 images (cross) and 35 images (longitudinal) respectively. They are shown below. (Fig. 8)

Fig. 8 (a)Cross section images (b)Longitudinal section images

We appreciate Yoko Kayama in Terabase for the kind assists related to TEM observation. Table 4 shows the condition of the observation.

Table 4 Conditions of TEM imaging

Magnification	30K
Resolution	4096×4096 pixels
The length of one side	1333.68nm

Materials

Each material was purchased from companies below. (Table 5)

Table 5 Materials

Material	Company
Agarose	TAKARA, JAPAN
M13mp18	TAKARA, JAPAN
SYBR gold	TAKARA, JAPAN
50xTAE	Wako Pure Chemical Industries, JAPAN
6xloading buffer	TAKARA, JAPAN
Staples	Eurofins Genomics, JAPAN

Equipment

Each equipment was purchased from companies below. (Table 6)

Table 6 Equipment

Equipment	Company
Mupid-2plus(Electrophoresis chamber)	Mupid, JAPAN
ChemiDoc MP (gel imager)	BIO-RAD, USA
Mastercycler pernonal (thermal cycler)	Eppendorf, USA
Nano Live Vision (AFM)	RIBM, JAPAN
Mica	Furuuchi Chemical Corporation, JAPAN
Micro Cantilever	OLYMPUS, JAPAN