Competitive annealing of multiple DNA origami: formation of chimeric origami

Abstract

Scaffolded DNA origami are a robust tool for building discrete nanoscale objects at high yield. This strategy ensures, in the design process, that the desired nanostructure is the minimum free energy state for the designed set of DNA sequences. Despite aiming for the minimum free energy structure, the folding process which leads to that conformation is difficult to characterize, although it has been the subject of much research. In order to shed light on the molecular folding pathways, this study intentionally frustrates the folding process of these systems by simultaneously annealing the staple pools for multiple target or parent origami structures, forcing competition. A surprising result of these competitive, simultaneous anneals is the formation of chimeric DNA origami which inherit structural regions from both parent origami. By comparing the regions inherited from the parent origami, relative stability of substructures were compared. This allowed examination of the folding process with typical characterization techniques and materials. Anneal curves were then used as a means to rapidly generate a phase diagram of anticipated behavior as a function of staple excess and parent staple ratio. This initial study shows that competitive anneals provide an exciting way to create diverse new nanostructures and may be used to examine the relative stability of various structural motifs.The commercial development of inexpensive and quickly produced DNA of arbitrary nucleobase sequence has fueled the growth of DNA nanotechnology as a field [1]. DNA nanotechnology has made promising advances toward light harvesting [2], computation [3], cancer treatement [4,5], and assembly of nanoelectronics [6]. While many DNA self-assembly strategies exist [7,8], scaffolded DNA origami has received significant attention as a convenient way to design and create discrete nanoscale objects [9]. Scaffolded DNA origami consist of single strand DNA (ssDNA) of two types, synthetic oligomers and circular viral genomes; the viral scaffold is forced to route through the designed structure by the binding of complementary subsequences on the synthetic oligomers, or staples. Staples are added in excess, often 10× relative to the scaffold concentration, strongly driving the viral ssDNA scaffold to fold into the target structure. Although 10× is a common staple excess anneals have been successfully performed as low as 2.5× staple excess. As this study involves multiple staple pools, staple excess will always refer to the total staple concentration relative to the scaffold, while the parent staple ratio will refer to how much of that total corresponds to the staple pool for each target origami. High production yield is achieved by thermal annealing and slow cooling of the system. Such annealing has also been performed chemically, isothermally, and mechanically [10][11][12].The hybridization of double strand DNA (dsDNA) has been well studied, particularly under physiological conditions [13,14]. The formation of dsDNA from ssDNA is driven by base stack...

Entropic effects in cell lineage tree packings

“… Optimal packings [ 1 , 2 ] of unconnected objects have been studied for centuries [ 3 – 6 ], but the packing principles of linked objects, such as topologically complex polymers [ 7 , 8 ] or cell lineages [ 9 , 10 ], are yet to be fully explored. Here, we identify and investigate a generic class of geometrically frustrated tree packing problems, arising during the initial stages of animal development when interconnected cells assemble within a convex enclosure [ 10 ].…”

“…Since Kepler’s 17th century conjecture on dense sphere packings [ 3 ], a rich body of experimental and theoretical work [ 19 ] has helped clarify the physical [ 20 ] and mathematical [ 1 , 2 ] principles underlying optimal embeddings of unconnected objects [ 5 , 6 , 21 ]. By contrast, much less is known about the packings of topologically linked structures in confined spaces, a setting relevant to the understanding of chromosome condensation [ 8 ], protein folding [ 22 ], multiscaffold DNA-origami structures [ 7 ], synthetic multicellular self-assembly [ 23 ] and the positioning of cell lineage- trees (CLTs) in embryonic cysts [ 10 , 24 ].…”

“…In particular, the presence of topological links arising from incomplete cytokinesis suggests that cell ordering and rearrangement processes can deviate strongly from the soap bubble paradigm used to describe patterns of organization of simple cell aggregates [ 12 ], with potentially profound implications for tissue scale organization and dynamics. More broadly, the insights from this study can provide useful guidance for the controlled self-assembly of topologically linked micro-structures in confined geometries, as realizable with modern DNA origami [ 7 ] and recently developed molecular linker toolboxes for multicellular self-assembly [ 23 ].…”

Insights into the Structure and Energy of DNA Nanoassemblies

“…Recent studies show that the beneficial effect of magnesium ions is largely dependent on the DNA superstructure and that, in some cases, those structures can survive in almost pure water for long times [92]. Only a few examples of DNA nanostructures have been reported in the presence of alternative buffers, such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) [68], cacodylate [93] or phosphate-buffered saline (PBS) [74]. The structural reconfiguration of a DNA origami structure from one state (red) to another (green) is mechanically triggered by the addition of staples that target the edges of the shape, passing through intermediate species (orange) along a folding landscape.…”

“…Recent studies show that the beneficial effect of magnesium ions is largely dependent on the DNA superstructure and that, in some cases, those structures can survive in almost pure water for long times [ 92 ]. Only a few examples of DNA nanostructures have been reported in the presence of alternative buffers, such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) [ 68 ], cacodylate [ 93 ] or phosphate-buffered saline (PBS) [ 74 ].…”