Switchable DNA-origami nanostructures that respond to their environment and their applications

Structural DNA nanotechnology, in which Watson-Crick base pairing drives the formation of self-assembling nanostructures, has rapidly expanded in complexity and functionality since its inception in 1981. DNA nanostructures can now be made in arbitrary three-dimensional shapes and used to scaffold many other functional molecules such as proteins, metallic nanoparticles, polymers, fluorescent dyes and small molecules. In parallel, the field of dynamic DNA nanotechnology has built DNA circuits, motors and switches. More recently, these two areas have begun to merge—to produce switchable DNA nanostructures, which change state in response to their environment. In this review, we summarise switchable DNA nanostructures into two major classes based on response type: molecular actuation triggered by local chemical changes such as pH or concentration and external actuation driven by light, electric or magnetic fields. While molecular actuation has been well explored, external actuation of DNA nanostructures is a relatively new area that allows for the remote control of nanoscale devices. We discuss recent applications for DNA nanostructures where switching is used to perform specific functions—such as opening a capsule to deliver a molecular payload to a target cell. We then discuss challenges and future directions towards achieving synthetic nanomachines with complexity on the level of the protein machinery in living cells.This work was supported by Australian Research Council Discovery Early Career Research Fellowship DE180101635 (SW), University of Sydney Nano Institute Scholarship (JKDS, MTL)

Complex multicomponent patterns rendered on a 3D DNA-barrel pegboard

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of similar to 90nm diameter and similar to 250nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of similar to 8nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology

LIPIcs, Volume 238, DNA 28, Complete Volume

LIPIcs, Volume 238, DNA 28, Complete Volum

Front Matter, Table of Contents, Preface, Conference Organization

Front Matter, Table of Contents, Preface, Conference Organizatio

Addressing the Instability of DNA Nanostructures in Tissue Culture

DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using <i>in vitro</i> tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced <i>via</i> the origami method to the <i>in vitro</i> tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO₄ prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg²⁺-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg²⁺ to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with <i>in vitro</i> tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two <i>in vitro</i> assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable experimental data. Our study thus describes considerations that are vital for researchers undertaking <i>in vitro</i> tissue culture studies with DNA nanostructures and some potential solutions for ensuring that nanostructure integrity and functions are maintained during experiments

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

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 &plusmn; 0.2% to 100 &plusmn; 2%), optimising the separation between adjacent cholesterols (64 &plusmn; 26% to 78 &plusmn; 30%), decreasing spacer length (38 &plusmn; 20% to 95 &plusmn; 5%), and using protective ssDNA 10T overhangs (38 &plusmn; 20% to 87 &plusmn; 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 &plusmn; 22% to 92 &plusmn; 12%), but samples re-aggregated after detergent removal (74 &plusmn; 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

Exaggeration and suppression of iridescence: the evolution of two-dimensional butterfly structural colours

Many butterfly species possess ‘structural’ colour, where colour is due to optical microstructures found in the wing scales. A number of such structures have been identified in butterfly scales, including three variations on a simple multi-layer structure. In this study, we optically characterize examples of all three types of multi-layer structure, as found in 10 species. The optical mechanism of the suppression and exaggeration of the angle-dependent optical properties (iridescence) of these structures is described. In addition, we consider the phylogeny of the butterflies, and are thus able to relate the optical properties of the structures to their evolutionary development. By applying two different types of analysis, the mechanism of adaptation is addressed. A simple parsimony analysis, in which all evolutionary changes are given an equal weighting, suggests convergent evolution of one structure. A Dollo parsimony analysis, in which the evolutionary ‘cost’ of losing a structure is less than that of gaining it, implies that ‘latent’ structures can be reused

A DNA-based molecular motor that can navigate a network of tracks.

Synthetic molecular motors can be fuelled by the hydrolysis or hybridization of DNA. Such motors can move autonomously and programmably, and long-range transport has been observed on linear tracks. It has also been shown that DNA systems can compute. Here, we report a synthetic DNA-based system that integrates long-range transport and information processing. We show that the path of a motor through a network of tracks containing four possible routes can be programmed using instructions that are added externally or carried by the motor itself. When external control is used we find that 87% of the motors follow the correct path, and when internal control is used 71% of the motors follow the correct path. Programmable motion will allow the development of computing networks, molecular systems that can sort and process cargoes according to instructions that they carry, and assembly lines that can be reconfigured dynamically in response to changing demands