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.
Highlights
Over the past 15 years, the development of DNA origami technology led to huge advances in the field of structural DNA nanotechnology, as it allows straightforward construction of large and complex nanostructures.[1]
Recent examples of DNA origami nanostructures designed in our lab include benchmark targets for single-molecule method development,[7] curved nanostructures to deform membranes,[8] or nanostructures serving as passive cargo to study transport processes in reaction−diffusion systems.[9]
We reasoned that the use of a well-characterized modular structure would be most convenient and opted for a flat rectangular grid origami used in a number of previous single-molecule fluorescence studies.[7,25,29,33,34]
Summary
Over the past 15 years, the development of DNA origami technology led to huge advances in the field of structural DNA nanotechnology, as it allows straightforward construction of large and complex nanostructures.[1]. One option is multivalent binding between origami monomers to facilitate nucleation.[20,24] for origami in 2D systems, increasing DNA origami monomer diffusion coefficients by adding monovalent cations and/or depositing particles on a fluid lipid bilayer rather than on a solid support accelerates assembly.[18,19,23] Additional acceleration comes from precisely matched and rigid geometries of the associating staple extensions to accelerate transition from monovalent binding nucleation to multivalent full binding.[20] Importantly, at least in solution, association rates for DNA origami dimerization reach values comparable to typical association rates for free DNA oligonucleotides.[20] This indicates that increasing effective association rates of the hybridization reaction itself may yield an additional gain in DNA origami superstructure assembly speed With this idea in mind, we reasoned that recent developments toward increasing hybridization on-rates in DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy could be transferred to accelerate DNA origami superstructure assembly.[25]. Our results provide useful insights for future experiments that require rapid cross-linking of DNA origami superstructures
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