Membrane compartmentalization and growth are central aspects of living cells, and are thus encoded in every cell’s genome. For the creation of artificial cellular systems, genetic information and production of membrane building blocks will need to be coupled in a similar manner. However, natural biochemical reaction networks and membrane building blocks are notoriously difficult to implement in vitro. Here, we utilized amphiphilic elastin-like peptides (ELP) to create self-assembled vesicular structures of about 200 nm diameter. In order to genetically encode the growth of these vesicles, we encapsulate a cell-free transcription-translation system together with the DNA template inside the peptide vesicles. We show in vesiculo production of a functioning fluorescent RNA aptamer and a fluorescent protein. Furthermore, we implement in situ expression of the membrane peptide itself and finally demonstrate autonomous vesicle growth due to the incorporation of this ELP into the membrane.
A light-driven artificial molecular nanomachine was constructed based on DNA scaffolding. Pyrene-modified walking strands and disulfide bond-connected stator strands, employed as anchorage sites to support walker movement, were assembled into a 2D DNA tile. Pyrene molecules excited by photoirradiation at 350 nm induced cleavage of disulfide bond-connected stator strands, enabling the DNA walker to migrate from one cleaved stator to the next on the DNA tile. The time-dependent movement of the walker was observed and the entire walking process of the walker was characterized by distribution of the walker-stator duplex at four anchorage sites on the tile under different irradiation times. Importantly, the light-fuelled mechanical movements on DNA tile were first visualized in real time during UV irradiation using high-speed atomic force microscopy (HS-AFM).
DNA‐binding proteins are promising reagents for the sequence‐specific modification of DNA‐based nanostructures. Here, we investigate the utility of a series of relaxase proteins—TrwC, TraI, and MobA—for nanofunctionalization. Relaxases are involved in the conjugative transfer of plasmids between bacteria, and bind to their DNA target sites via a covalent phosphotyrosine linkage. We study the binding of the relaxases to two standard DNA origami structures—rodlike six‐helix bundles and flat rectangular origami sheets. We find highly orthogonal binding of the proteins with binding yields of 40–50 % per binding site, which is comparable to other functionalization methods. The yields differ for the two origami structures and also depend on the position of the binding sites. Due to their specificity for a single‐stranded DNA target, their orthogonality, and their binding properties, relaxases are a uniquely useful addition to the toolbox available for the modification of DNA nanostructures with proteins.
Dynamic DNA nanodevices are designed to perform structure‐encoded motion actuated by a variety of different physicochemical stimuli. In this context, hybrid devices utilizing other components than DNA have the potential to considerably expand the library of functionalities. Here, the reversible reconfiguration of a DNA origami structure using the stimulus sensitivity of elastin‐like polypeptides is reported. To this end, a rectangular sheet made using the DNA origami technique is functionalized with these peptides and by applying changes in salt concentration the hydrophilic–hydrophobic phase transition of these peptides actuate the folding of the structure. The on‐demand and reversible switching of the rectangle is driven by externally imposed temperature oscillations and appears at specific transition temperatures. Using transmission electron microscopy, it is shown that the structure exhibits distinct conformational states with different occupation probabilities, which are dependent on structure‐intrinsic parameters such as the local number and the arrangement of the peptides on the rectangle. It is also shown through ensemble fluorescence resonance energy transfer spectroscopy that the transition temperature and thus the thermodynamics of the rectangle‐peptide system depends on the stimuli salt concentration and temperature, as well as on the intrinsic parameters.
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