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DNA tethers

The idea behind vertical DNA origami stacks is described in the Idea section (vertical stacks) and their potential applications for nanoelectronics in the Discussion. Vertical DNA origami stacks could be fabricated based on either DNA or protein tethers.

The idea behind DNA tethers is to incorporate unique complementary single stranded sequences protruding perpendicularly from the DNA origami, where their positions on the opposing face of the other DNA origami stacking partner was decorated at the mirroring positions (Figure 32). Since we created a tube-like structure in the initial attempt when DNA tethers were positioned only at the two long sides of the rectangle we decided to use tethering at 10 positions that are distributed asymmetrically throughout the surface of the DNA rectangle. We designed ten unique tethering sequences. In our design selected standard staples were replaced with the corresponding staples that were elongated with the appropriate single stranded binding sequences. We separately annealed two types of rectangles that contained the first set and the second complementary set of binding sequences. Each type was ligated with T4 DNA ligase overnight at 16 °C to increase the DNA origami stability. After ligation we removed the excess staple strands with filtration. Then we mixed the two DNA rectangles as depicted in Figure 32 at the 1:1 ratio.

Figure 32: Schematic representation of the idea and implementation of DNA tethers for vertical DNA origami stacks. a. creation of two complementary DNA origami rectangles decorated with complementary single-stranded tethers; b. asymmetric arrangement of 10 tethering positions on two DNA origami rectangles.
We designed 10 unique binding sequences for DNA origami rectangle stacking. Set 1 comprises standard staples that were extended with appropriate binding sequences. To each staple a binding sequence at their 3'-terminus was added except to staples CS4 and ES4 (see Methods/DNA Origami design/Modifications). Set 2 comprises binding staples composed of 10 pairs of half staples. The first half staple contains the first half of the sequence from the original staple and is elongated with appropriate complementary binding sequence according to its position at its 3'-terminus. The second half staple contains the rest of the original staple sequence. This separation was performed so that binding sequences would protrude from the rectangle at the opposite face of the layer while preserving the orientation.

We divided the mix into aliquots and heated them to 75, 65, 55 and 45 °C respectively. The mixtures were allowed to cool with the rate of -1 °C/min. AFM analysis of samples revealed total DNA origami degradation of samples heated to 75 and 65 °C. DNA rectangles were present in the sample heated up to 55 °C, but their structure was severely damaged, and no stacks were observed.
Figure 33: a) AFM imaging of DNA origami rectangles annealed at different temperatures. Complementary DNA rectangles were mixed and reannealed at 55 (a), 65 (b) and 75°C (c) to anneal DNA tethers and form stacks. Although rectangular structures are easily discernible, heating the sample to 55 °C already visibly damages the fold. b) and c) demonstrate the complete collapse of DNA origami structures upon heating to 65 and 75 °C respectively.

Next we tested the same approach using non-ligated DNA rectangles with an excess of the remaining staple strands. We reasoned that during the heating process some staple strands might detach from long M13 single stranded DNA and destabilize the fold. Presence of the excess staples allows them to replace the detached staples. We separately annealed two different sets of DNA origami as before, mixed them at 1:1 ratio and divided into aliquots. We then heated the aliquots to 50, 55, 60, 65 and 70 °C. Although yields were not very high, we found origami stacks in all of the samples. Origami stacks are recognizable as brighter rectangles that showed twice the height of the majority of rectangles which is clearly seen on the height profile.

Figure 34: Formation of DNA origami stacks with the expected height visualized by the AFM. The upper left sample was annealed from 70 °C and the lower from 60 °C. Both samples contained perfectly arranged double layer stacks. Red arrows indicate the zigzag path of the profile extraction made with PicoImage software (Agilent). Profile clearly depicts that the height of stacks is double in size of the non-stacked DNA origami rectangles.

Our results demonstrate that vertical DNA stacks can be prepared using DNA tethers and that we can produce a perfect superposition of the two DNA rectangles. Length of the DNA tether used was 20 nucleotides, which means that the maximal distance between the stacks could be around 7 nm. However in our samples the DNA was dehydrated and the DNA origami stack height collapsed to approximately double height of the single DNA rectangle. Presumably in aqueous solution the distance should depend on the ionic strength and presence of ions, however for the fixed separation of the stacks protein tethers/spacers clearly represent a better choice.

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