Design Features to Accelerate the Higher-Order Assembly of DNA Origami on Membranes

Abstract

Nanotechnology often exploits DNA origami nanostructures assembled into even larger superstructures up to micrometer sizes with nanometer shape precision. However, large-scale assembly of such structures is very time-consuming. Here, we investigated the efficiency of superstructure assembly on surfaces using indirect cross-linking through low-complexity connector strands binding staple strand extensions, instead of connector strands binding to scaffold loops. Using single-molecule imaging techniques, including fluorescence microscopy and atomic force microscopy, we show that low sequence complexity connector strands allow formation of DNA origami superstructures on lipid membranes, with an order-of-magnitude enhancement in the assembly speed of superstructures. A number of effects, including suppression of DNA hairpin formation, high local effective binding site concentration, and multivalency are proposed to contribute to the acceleration. Thus, the use of low-complexity sequences for DNA origami higher-order assembly offers a very simple but efficient way of improving throughput in DNA origami design.

Inducing Lipid Domains in Membranes by Self‐Assembly of DNA Origami

“…The anchored DNA origami rapidly diffuses in its monomeric form, but diffusion becomes much slower and a large fraction becomes even immobilized upon cross-linking by polyT crosslinkers. [19] This decrease in mobility further supports the formation of superstructures (Video S2, Supporting Information). The self-assembly of DNA origami decreases the diffusion coefficient of the anchored lipid molecules in the membrane (Figure S4, Supporting Information) from 1.6 ± 0.2 to 0.8 ± 0.3 µm 2 s −1 .…”

“…First, the rectangular one‐layer origami structure was adapted and improved from our previously reported design with some modifications. [ 19 ] DNA sequences and design features can be found in detail in Figure S1 and Table S1 of the Supporting Information. In particular, the design consists of 24 helices (≈70 × 100 nm) and is modified with more staples for increased binding strength to lipid surfaces through cholesterol antihandles, and additional staples to allow for the cross‐linking at desired sides of the nanostructure ( Figure a).…”

“…To gain maximum crosslinking efficiency without too much loss of specificity, four staples with polyA overhangs were placed at each side, which can cross-link with those of another origami using polyT connector strands. In comparison to previous structures, [ 19 ] the overhangs do not have the same directionality but are alternating, which is expected to increase the specificity. The staples used to attach fluorophores to the origami consist of a 19-nt overhang where the corresponding oligonucleotide modified with an imager could permanently bind.…”

“…DNA Origami Folding and Purification: The DNA origami structures were assembled as described before. [19,20] The single stranded scaffold DNA (p7249, tilibit nanosystems, 10 nm) was mixed with the staple oligonucleotides (eurofins genomics) in a 1:10 ratio in a folding buffer (1xTE, 5 mm NaCl) containing required amounts of MgCl 2 . For the rectangle the buffer consisted of 12.5 mm MgCl 2 and the folding reaction was performed using an initial 5 min melting step at 80 °C, followed by decreasing temperature ramp 60 to 4 °C over 3 h. By contrast, the folding reaction of the rod-shaped origami consisted of 20 mm MgCl 2 with an initial melting step 65 to 60 °C in 1 h and temperature ramp from 59 to 40 °C in 40 h. The structures were purified using a PEG-purification.…”

Protein‐Assisted Large‐Scale Assembly and Differential Patterning of DNA Origami Lattices

“…and Qutbuddin et al. [ 35,36 ] who used T linkers to crosslink DNA origami structures. To confirm that the lattices were indeed cross‐linked and stable after removal of the Min proteins, samples were washed with a buffer containing protein‐degrading agents (either proteinase K or guanidinium‐chloride), as well as Mg 2+ and K + (5 and 150 m m , respectively).…”