Binding of DNA origami to lipids: maximizing yield and switching via strand displacement

Singh, Jasleen; Darley, Esther; Ridone, Pietro; Gaston, James P.; Abbas, Ali; Wickham, Shelley F. J.; Baker, Matthew A. B.

doi:10.1093/nar/gkab888

Cited by 26 publications

(26 citation statements)

References 85 publications

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“…For the proximal spacer design, a high yield was observed for all configurations, with yields in the range of 90-95% for the circular configurations and 95-101% for the rectangular configurations (Figure 2B(i)), suggesting minimal aggregation. This was expected based on previous studies, which show low aggregation for proximal placement [14] but also lower membrane-binding yield [37]. Note that a yield of over 100% is possible as these yields are normalized to the control lane, and gel loading and analysis error can be up to 10% RSD [47].…”

Section: Spacersupporting

confidence: 74%

“…The DOT is a flexible single-layered 24-helix rectangular 2D DNA nanostructure (60 × 90 nm), while the DOB is a barrel-shaped rigid 3D structure (52 × 30 nm). In a previous study, we showed that increasing the number of cholesterols does not always increase the membrane binding, and that the optimum number of cholesterols for the DOT structure is 4-8 [37]. Here, we compare the aggregation for two different configurations of cholesterols on the DOT: a fourcholesterol rectangular (R) configuration and a six-cholesterol circular (C) configuration.…”

Section: Introductionmentioning

confidence: 90%

“…and that the optimum number of cholesterols for the DOT structure is 4-8 [37]. Here, we compare the aggregation for two different configurations of cholesterols on the DOT: a four-cholesterol rectangular (R) configuration and a six-cholesterol circular (C) configuration.…”

Section: The Design and Folding Of Dna Origami Nanostructuresmentioning

confidence: 99%

“…The DOT was designed to be decorated by four and six cholesterols, in the rectangular and circular configurations, respectively. Four to six cholesterols were selected as we have previously shown that the membrane binding of the DOT is maximum when labelled with four to eight cholesterols [37]. Due to the staple nick pattern of the DOT, six instead of eight cholesterols were selected in the circular configuration to allow for even separation between the cholesterol groups.…”

Section: Effect Of Number and Separation Of Cholesterols Spacer Lengt...mentioning

confidence: 99%

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Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures

et al. 2021

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DNA nanotechnology provides methods for building custom membrane-interacting nanostructures with diverse functions, such as shaping membranes, tethering defined numbers of membrane proteins, and transmembrane nanopores. The modification of DNA nanostructures with hydrophobic groups, such as cholesterol, is required to facilitate membrane interactions. However, cholesterol-induced aggregation of DNA origami nanostructures remains a challenge. Aggregation can result in reduced assembly yield, defective structures, and the inhibition of membrane interaction. Here, we quantify the assembly yield of two cholesterol-modified DNA origami nanostructures: a 2D DNA origami tile (DOT) and a 3D DNA origami barrel (DOB), by gel electrophoresis. We found that the DOT assembly yield (relative to the no cholesterol control) could be maximised by reducing the number of cholesterols from 6 to 1 (2 ± 0.2% to 100 ± 2%), optimising the separation between adjacent cholesterols (64 ± 26% to 78 ± 30%), decreasing spacer length (38 ± 20% to 95 ± 5%), and using protective ssDNA 10T overhangs (38 ± 20% to 87 ± 6%). Two-step folding protocols for the DOB, where cholesterol strands are added in a second step, did not improve the yield. Detergent improved the yield of distal cholesterol configurations (26 ± 22% to 92 ± 12%), but samples re-aggregated after detergent removal (74 ± 3%). Finally, we confirmed functional membrane binding of the cholesterol-modified nanostructures. These findings provide fundamental guidelines to reducing the cholesterol-induced aggregation of membrane-interacting 2D and 3D DNA origami nanostructures, improving the yield of well-formed structures to facilitate future applications in nanomedicine and biophysics.

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Section: Spacersupporting

confidence: 74%

Section: Introductionmentioning

confidence: 90%

Section: The Design and Folding Of Dna Origami Nanostructuresmentioning

confidence: 99%

Section: Effect Of Number and Separation Of Cholesterols Spacer Lengt...mentioning

confidence: 99%

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Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures

et al. 2021

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“…Importantly, it was reported that aggregation lowers the membrane insertion rate of DNA structures [20,47], and our observations showed similar effects even for shielded structures and in the presence of oPOE surfactants. Thus, various methods have been employed to minimize the impact of clustering [33]: optimizing the separation between cholesterols and decreasing the length of a spacer, folding in the presence of the surfactant [27,31,33], adding cholesterol in the second folding step [24,25,33], and, finally, the shielding technique used in this work [22,33,48,49].…”

Section: Discussionmentioning

confidence: 99%

A Surfactant Enables Efficient Membrane Spanning by Non-Aggregating DNA-Based Ion Channels

2022

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DNA nanotechnology makes use of hydrophobically modified constructs to create synthetic membrane protein mimics. However, nucleic acid structures exhibit poor insertion efficiency, leading to a low activity of membrane-spanning DNA protein mimics. It is suggested that non-ionic surfactants improve insertion efficiency, partly by disrupting hydrophobicity-mediated clusters. Here, we employed confocal microscopy and single-molecule transmembrane current measurements to assess the effects of the non-ionic surfactant octylpolyoxyethylene (oPOE) on the clustering behavior and membrane activity of cholesterol-modified DNA nanostructures. Our findings uncover the role of aggregation in preventing bilayer interactions of hydrophobically decorated constructs, and we highlight that premixing DNA structures with the surfactant does not disrupt the cholesterol-mediated aggregates. However, we observed the surfactant’s strong insertion-facilitating effect, particularly when introduced to the sample separately from DNA. Critically, we report a highly efficient membrane-spanning DNA construct from combining a non-aggregating design with the addition of the oPOE surfactant.

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Engineering DNA Nanostructures to Manipulate Immune Receptor Signaling and Immune Cell Fates

Tseng

Wang

Douglas

et al. 2021

Adv Healthcare Materials

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Immune cells sense, communicate, and logically integrate a multitude of environmental signals to make important cell-fate decisions and fulfill their effector functions. These processes are initiated and regulated by a diverse array of immune receptors and via their dynamic spatiotemporal organization upon ligand binding. Given the widespread relevance of the immune system to health and disease, there have been significant efforts toward understanding the biophysical principles governing immune receptor signaling and activation, as well as the development of biomaterials which exploit these principles for therapeutic immune engineering. Here, how advances in the field of DNA nanotechnology constitute a growing toolbox for further pursuit of these endeavors is discussed. Key cellular players involved in the induction of immunity against pathogens or diseased cells are first summarized. How the ability to design DNA nanostructures with custom shapes, dynamics, and with site-specific incorporation of diverse guests can be leveraged to manipulate the signaling pathways that regulate these processes is then presented. It is followed by highlighting emerging applications of DNA nanotechnology at the crossroads of immune engineering, such as in vitro reconstitution platforms, vaccines, and adjuvant delivery systems. Finally, outstanding questions that remain for further advancing immune-modulatory DNA nanodevices are outlined.

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Binding of DNA origami to lipids: maximizing yield and switching via strand displacement

Cited by 26 publications

References 85 publications

Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures

Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures

A Surfactant Enables Efficient Membrane Spanning by Non-Aggregating DNA-Based Ion Channels

Engineering DNA Nanostructures to Manipulate Immune Receptor Signaling and Immune Cell Fates

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