Shelley Wickham

Direct observation of stepwise movement of a synthetic molecular transporter

Controlled motion at the nanoscale can be achieved by using Watson-Crick base-pairing to direct the assembly and operation of a molecular transport system consisting of a track, a motor and fuel, all made from DNA. Here, we assemble a 100-nm-long DNA track on a two-dimensional scaffold, and show that a DNA motor loaded at one end of the track moves autonomously and at a constant average speed along the full length of the track, a journey comprising 16 consecutive steps for the motor. Real-time atomic force microscopy allows direct observation of individual steps of a single motor, revealing mechanistic details of its operation. This precisely controlled, long-range transport could lead to the development of systems that could be programmed and routed by instructions encoded in the nucleotide sequences of the track and motor. Such systems might be used to create molecular assembly lines modelled on the ribosome.

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.

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 in vitro 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 via the origami method to the in vitro 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 in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro 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 in vitro tissue culture studies with DNA nanostructures and some potential solutions for ensuring that nanostructure integrity and functions are maintained during experiments.

The chiral structure of porous chitin within the wing-scales of Callophrys rubi

The structure of the porous three-dimensional reticulated pattern in the wing scales of the butterfly Callophrys rubi (the Green Hairstreak) is explored in detail, via scanning and transmission electron microscopy. A full 3D tomographic reconstruction of a section of this material reveals that the predominantly chitin material is assembled in the wing scale to form a structure whose geometry bears a remarkable correspondence to the srs net, well-known in solid state chemistry and soft materials science. The porous solid is bounded to an excellent approximation by a parallel surface to the Gyroid, a three-periodic minimal surface with cubic crystallographic symmetry I4₁32, as foreshadowed by Stavenga and Michielson. The scale of the structure is commensurate with the wavelength of visible light, with an edge of the conventional cubic unit cell of the parallel-Gyroid of approximately 310 nm. The genesis of this structure is discussed, and we suggest it affords a remarkable example of templating of a chiral material via soft matter, analogous to the formation of mesoporous silica via surfactant assemblies in solution. In the butterfly, the templating is achieved by the lipid-protein membranes within the smooth endoplasmic reticulum (while it remains in the chrysalis), that likely form cubic membranes, folded according to the form of the Gyroid. The subsequent formation of the chiral hard chitin framework is suggested to be driven by the gradual polymerisation of the chitin precursors, whose inherent chiral assembly in solution (during growth) promotes the formation of a single enantiomer.

Routing of individual polymers in designed patterns

Synthetic polymers are ubiquitous in the modern world, but our ability to exert control over the molecular conformation of individual polymers is very limited. In particular, although the programmable self-assembly of oligonucleotides and proteins into artificial nanostructures has been demonstrated, we currently lack the tools to handle other types of synthetic polymers individually and thus the ability to utilize and study their single-molecule properties. Here we show that synthetic polymer wires containing short oligonucleotides that extend from each repeat can be made to assemble into arbitrary routings. The wires, which can be more than 200 nm in length, are soft and bendable, and the DNA strands allow individual polymers to self-assemble into predesigned routings on both two- and three-dimensional DNA origami templates. The polymers are conjugated and potentially conducting, and could therefore be used to create molecular-scale electronic or optical wires in arbitrary geometries.

Iridescence from photonic crystals and its suppression in butterfly scales

Regular three-dimensional periodic structures have been observed in the scales of over half a dozen butterfly species. We compare several of these structures: we calculate their photonic bandgap properties; measure the angular variation of the reflection spectra; and relate the observed iridescence (or its suppression) to the structures. We compare the mechanisms for iridescence suppression in different species and conclude with some speculations about form, function, development and evolution.

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 programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads

Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.

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.

Control of iridescence in natural photonic structures: the case of butterfly scales

Abstract: This chapter looks at how optical microstructures in butterfly scales produce colour, as well as structural black and white. Butterflies contain some of the most diverse optical microstructures nature has to offer. The most interesting and complex of these are three-dimensional photonic crystals. The refractive index contrasts available in butterflies mean that complete band gaps (implying the structure retains its colour from all angles) are impossible. In this context, the role of chirality, composite structures, and randomness are particularly interesting. We also consider how structures in butterfly scales can exaggerate iridescence or, conversely, produce structures that either reflect or absorb across the whole visible spectrum.