Protocol

　
Design of the DNA hydrogel

st1: 5’- TGGATCCAGTTTGTTATCGCAGGAGCGTCGGTATTCAAA -3’
st2: 5’- TGGATCCAGTTTGAATACCGACGCCACGACCTAATCTTA -3’
st3: 5’- TGGATCCAGTAAGATTAGGTCGTGATGGTGAAATGTAAA -3’
st4: 5’- TGGATCCAGTTTACATTTCACCATTCCTGCGATAACAAA -3’
st2 t8   : 5’- CACTCTTCTGGATCCAGTTTGAATACCGACGCCACGACCTAATCTTA -3’
(st2 t8)*: 5’- TAAGATTAGGTCGTGGCGTCGGTATTCAAACTGGATCCAGAAGAGTG -3’
st4 t8   : 5’- CACTCTTCTGAATCCAGTTTACATTTCACCATTCCTGCGATAACAAA -3’
(st4 t8)*: 5’- TTTGTTATCGCAGGAATGGTGAAATGTAAACTGGATCCAGAAGAGTG -3’
Fig.3:(A) Basic DNA hydrogel motif. (B) The motif made by st1, st2 t8, st3, st4 t8 and complimentary strand to st2 t8, st4 t8. (C) The structure after annealed together.

1, Experiment to see if the gel has formed

We checked if the gel has formed by following the same protocol as the reference experiment.

Mix

　 　　 　　 　 　 　　 　　 　 　 　　 　　 　

Table.1: The final concentration of the solution used in experiment 1. *TE(pH 8.0): 10 mM Tris pH8.0, 1 mM EPTA pH8.0

	control	gel
TE(pH 8.0)*	1x	1x
NaCl	50 mM	50 mM
DNA strands	0 μM	200 μM

PCR
The Condition of Annealing in experiment 1 is shown in Fig.4

Fig.4: Annealing condition. DNA strands are annealed by cooling from 95 to 4°C in 1.5 h.

2, Experiment to find the optimal DNA concentration

We decided the optimal concentration of the DNA strands.

Mix

　 　 　 　　 　　 　 　 　　 　　 　

Table.2: Summary of the experiment 2 condition. The final concentration of the each solution is shown in the table.

	Tube1	Tube2	Tube3	Tube4
TE(pH 8.0)	1x	1x	1x	1x
NaCl	50 mM	50 mM	50 mM	50 mM
DNA strands	0 μM	50 μM	75 μM	100 μM

PCR
Annealing condition was the same as experiment 1 (shown in Fig.4).

3,Experiments to check the state of gel by changing combination of sequences

We tested how the gel state varies with a combination of DNA sequences.
Table.3: Summary of the DNA sequences combination. The image is the expected structure after PCR.

Mix

　 　 　 　　 　　 　 　　 　　 　 　　 　　 　

Table.4: The final concentration of the solution used in experiment 3.

Solution	Final concentration
TE(pH 8.0)	1x
DNA strands	100 μM
NaCl	50 mM

Annealing condition
After mixing solutions, PCR was sat in the condition shown in Fig.5.

Fig.5: Annealing condition in experiment 3. After denaturation at 95°C for 10 min, the temperature was reduced to 25°C at intervals of 1°C per 14 seconds.

4, Experiments to check the state of gel by adding complementary strand of st2 t8 and st4 t8

4-1, only st2 t8

We tried to create the motif by replacing st2 and st4 to st2 t8 and st4 t8 which we designed, and checked the gel state are changed or not with adding (st2 t8)* and (st4 t8)* which strands are complementarily binding to st2 t8 and st4 t8. First, we tried with swapping st2 to st2 t8 and added (st2 t8)*.
Fig.6: (A)The motif made by st1, st2 t8, st3, st4 t8. (B)The image of the expected structure after PCR.
Table.5: Condition of experiment 4-1.

Mix

　 　 　 　　 　　 　 　　 　　 　 　　 　　 　 　　 　　 　

Table.6: The final concentration of the solution used in experiment 4-1.

Solution	Final concentration
TE(pH 8.0)	1x
DNA strands	100 μM
Complementary strand	200 μM
NaCl	50 mM

Annealing condition
The setting of PCR was the same as experiment 3 (shown in Fig.5).

4-2, st2 t8 and st4 t8

After that, we confirmed that the gel was formed or not with adding (st4 t8)* complementary to st4 t8 which in the new motif.
Fig.7: (A)The motif build by st1, st2 t8, st3 and st4 t8. (B)The image of the expected structure after PCR.
Table.7: Condition of experiment 4-2.

Mix

　 　 　 　　 　　 　 　　 　　 　 　　 　　 　

Table.8: The final concentration of the solution used in experiment 4-2.

Solution	Final concentration
TE(pH 8.0)	1x
DNA strands	100 μM
Complementary strand	200 μM

Annealing condition
The protocol of PCR was the same as experiment 3 (shown in Fig.5).

Material

TE(pH 8.0) (Ambion,US)
10 mM Tris pH8.0
1 mM EPTA pH8.0
0.2 μm filtered

DNA strands (Integrated DNA Technologies, US)

Equipment

Rotor Gene™ Q Real-Time Rotary Analyzer (QIAGEN, Germany)
T319-4N 0.1 ml Tube and Cap Strips for Qiagen Rotor-Gene™ Q Real-Time Rotary Analyzer (QIAGEN, Germany)

Reference
[1] Kandatsu, D. et al. “Reversible Gel-Sol Transition of a Photo-Responsive DNA Gel.” Chem Bio Chem 17, 2016, 1118-1121