· Setting of folding patterns and number of bases · Design a simplified figure with ChemDraw · Adjustment of seam · Adjust seam positions not to overlap · Calculate the number of bases (32 units) between transfer points
· Using M13 of 7560 bases (Eurofin) · Design Y sequence by Adobe Illustrator CC 2018
· When creating in units of 32 bases, it exceeds 7560 bases. So we reduce each one base in the area surrounded by red and adjust them. · Fill in the sequence of 7560 bases (Eurofin)
· Fill in matched strand using matched strand script · Design a staple pattern
· It was corrected since the transfer position of the scaffold and the staple is shifted by a half pitch. As a result, we obtained a position where seam overlapped, so the shape of the staple pattern was modified not to be continuous · Attach staple number · Insert a sequence of hairpin between QR code position detection pattern and timing pattern (to emerge)
· Prepare staple DNA mix to design DNA QR code and mix it with M13mp18 ssDNA and 10 × TAE / Mg2＋buffer. Mess up with sterile water so that M13mp18 ssDNA becomes 4 nM and staple DNA mix becomes 20 nM, and annealing (90 ˚ C for 10 min, 90 ˚ C → 25 ˚ C, -1 ˚ C / min) and DNA QR make a sample of the code. Thereafter, do ultrafiltration (50 k Amicon 15 ° C., 10000 rpm 10 min × 3 times) Then, drop 1 μl of sample and 30 μl of 1 × TAE / Mg2＋buffer on mica and observe with AFM.
· This is the result of observing by AFM.
· As the shape of the QR code became a parallelogram instead of a square, we need to change the design of the QR code.
· We were able to observe that the height of the part which the dumbbell hairpin was inserted turned white.