Biomod/2014/Kansai/Protocols

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At first, we prepared staple DNA mixture of part of A. This mixture was kept at 90˚C for 10 minutes and cooled from 90˚C to 25˚C at a rate of -1.0˚C/min to anneal the strands. We observed it by AFM. Figure 1A and Figure 1B depict the AFM images at this condition.

We could observe rectangular structure and dumbbell hairpin. The ratio of length and breadth of the observed structure is about 1 : 3. We thought that we could observe a clear structure to slow an annealing rate. Accordingly, we switched it from -1.0°C/min to -0.1°C/min and observed the product by AFM. Figure 2A and Figure 2B depict the AFM images at slowly condition.

We could observe a structure, but it isn’t rectangular structure and is split structure. So, we thought that the formation of a structure isn’t influenced by an annealing rate.

In addition, we could observe clearly a structure at the first condition. Figure 3A and 3B depict the AFM images at this time. However, the dumbbell hairpin wasn’t observed. We suppose that a power of tapping by Cantilever is too strong and dumbbell hairpin has been destroyed.

To observe it more clear needs improvement of our observing skill by AFM. So, We confirmed whether three kinds of origami structure (A, B and C) is formed correctly by using agarose gel electrophoresis (1.5% agarose gel, 200 V, 400 mA, 60 min) . Figure 4 depicts the result of electrophoresis.

Lane1, 2 and 3 is part A, B and C of nano QR code. Two bands were observed in every lane. We annealed the origami that contained staple DNA of joints. There are possibilities that every origami form tube structure or multimers. We thought that this cause is connecting strands between A and B and between B and C are virtually same. So we excepted joints staples and confirmed whether origami structure is formed correctly by using agarose gel electrophoresis (1.5% agarose gel, 200 V, 400 mA, 60 min) . Figure 5 depicts the result of electrophoresis.

Since the band became single, we thought each origami (A, B, and C) is predictably formed. Therefore we created a sheet of origami connected by origami A and B and C.

Next, we considered the plan of connecting 3 kinds of origami. We have the problem that the sequences of 3 origami structures are very similar. Sequence of joints staples between A and B is much the same as that between B and C. Only difference of the two is the presence or absence of dumbbell hairpin. There was possibility that the joints staples between A and B connected origami B and C in error. It induces forming wrong structures (e.g. A-C) and multimeric. So, we needed to think seriously about how to prevent these problems. We thought two plans to connect 3 kinds of origami.

1

Figure 6 depicts the image of strategy of 1st plan. After each origami is formed, joints AB (1.2 eq.) and origami A (1.0 eq.) were mixed. By pumping the mixture (cooled from 37℃ to 25℃×3), we connected origami A with joints AB. After that, by adding origami B (1.0 eq.) and annealing these (kept at 37℃ for 10 minutes and cooled from 37℃ to 4℃), we connected origami A-joints AB with origami B. Next, by adding origami C (1.0 eq.) and joints BC (1.2 eq.) and annealing these (kept at 37℃ for 10 minutes and cooled from 37℃ to 4℃), we connected origami A-B with origami C. This is 1st plan.

2

Figure 7 depicts the image of strategy of 2nd plan. The way of connecting A-B of 1st plan and 2nd plan is same. On the other hand, joints BC (1.2 eq.) and origami C (1.0 eq.) were mixed. By pumping the mixture (cooled from 37℃ to 25℃×3), we connected origami C with joints BC. Finally, Origami A-B (1 eq.) and origami C-joints BC (1.0 eq.) were mixed. By annealing these (kept at 37℃ for 10 minutes and cooled from 37℃ to 4℃), we connected origami A-B with origami C. This is 2nd plan.

We confirmed whether 3 kinds of origami structures are connected correctly by using agarose gel electrophoresis (1.5% agarose gel, 200 V, 400 mA, 60 min). Figure 8 depicts the image of electrophoresis of two plans.

We observed the origami by AFM. But structure that height was 40-50 nm was only observed. We couldn’t observe the target.

We thought that it was because of multimerization by origami B. So we reduced the amount of origami B compared with origami A and origami C. We had experiment once again.

But there was no change in the result.

As solving the problem that sequence of joints staples between A and B is much the same as the sequence between B and C, we devised the idea. We filled new staple instead of excepted staples.

As a new way, after each origami is formed, we added joints staples between origami A and B into origami A. By pumping these, we connected origami A with the joints staples. Performing an ultrafiltration, we removed extra joints staples. After that we added origami B into it. By pumping these, we connected origami A with origami B. Now, in order to connect origami B reliably with origami A, we reduced the amount of origami B compared with origami A, we co. We added joints staples between origami B and C into it. By pumping these, we connected origami A and B with the joints staples. Performing an ultrafiltration, extra joints staples were removed. We added origami C into it. By pumping these, we connected origami A and origami B and origami C.