Choice of fluorophore affects dynamic DNA nanostructures

The ability to dynamically remodel DNA origami structures or functional nanodevices is highly desired in the field of DNA nanotechnology. Concomitantly, the use of fluorophores to track and validate the dynamics of such DNA-based architectures is commonplace and often unavoidable. It is therefore crucial to be aware of the side effects of popular fluorophores, which are often exchanged without considering the potential impact on the system. Here, we show that the choice of fluorophore can strongly affect the reconfiguration of DNA nanostructures. To this end, we encapsulate a triple-stranded DNA (tsDNA) into water-in-oil compartments and functionalize their periphery with a single-stranded DNA handle (ssDNA). Thus, the tsDNA can bind and unbind from the periphery by reversible opening of the triplex and subsequent strand displacement. Using a combination of experiments, molecular dynamics (MD) simulations, and reaction-diffusion modelling, we demonstrate for 12 different fluorophore combinations that it is possible to alter or even inhibit the DNA nanostructure formation—without changing the DNA sequence. Besides its immediate importance for the design of pH-responsive switches and fluorophore labelling, our work presents a strategy to precisely tune the energy landscape of dynamic DNA nanodevices

Nondeterministic self-assembly with asymmetric interactions.

We investigate general properties of nondeterministic self-assembly with asymmetric interactions, using a computational model and DNA tile assembly experiments. By contrasting symmetric and asymmetric interactions we show that the latter can lead to self-limiting cluster growth. Furthermore, by adjusting the relative abundance of self-assembly particles in a two-particle mixture, we are able to tune the final sizes of these clusters. We show that this is a fundamental property of asymmetric interactions, which has potential applications in bioengineering, and provides insights into the study of diseases caused by protein aggregation.Winton Programme for the Physics of Sustainability, Gates Cambridge, Oppenheimer PhD studentship, NanoDTC Cambridge (Grant ID: EP/L015978/1), Engineering and Physical Sciences Research Council (Grant ID: EP/L504920/1), Royal SocietyThis is the author accepted manuscript. The final version is available from the American Physical Society via http://dx.doi.org/10.1103/PhysRevE.94.02240

Bilayer-spanning DNA nanopores with voltage-switching between open and closed state.

Membrane-spanning nanopores from folded DNA are a recent example of biomimetic man-made nanostructures that can open up applications in biosensing, drug delivery, and nanofluidics. In this report, we generate a DNA nanopore based on the archetypal six-helix-bundle architecture and systematically characterize it via single-channel current recordings to address several fundamental scientific questions in this emerging field. We establish that the DNA pores exhibit two voltage-dependent conductance states. Low transmembrane voltages favor a stable high-conductance level, which corresponds to an unobstructed DNA pore. The expected inner width of the open channel is confirmed by measuring the conductance change as a function of poly(ethylene glycol) (PEG) size, whereby smaller PEGs are assumed to enter the pore. PEG sizing also clarifies that the main ion-conducting path runs through the membrane-spanning channel lumen as opposed to any proposed gap between the outer pore wall and the lipid bilayer. At higher voltages, the channel shows a main low-conductance state probably caused by electric-field-induced changes of the DNA pore in its conformation or orientation. This voltage-dependent switching between the open and closed states is observed with planar lipid bilayers as well as bilayers mounted on glass nanopipettes. These findings settle a discrepancy between two previously published conductances. By systematically exploring a large space of parameters and answering key questions, our report supports the development of DNA nanopores for nanobiotechnology.The SH lab is supported by the Leverhulme Trust (RPG-170), UCL Chemistry, EPSRC (Institutional Sponsorship Award), the National Physical Laboratory, and Oxford Nanopore Technologies. KG acknowledges funding from the Winton Program of Physics for Sustainability, Gates Cambridge and the Oppenheimer Trust. UFK was supported by an ERC starting grant #261101.This is the final version of the article. It was first published by ACS under the ACS AuthorChoice license at http://dx.doi.org/10.1021/nn5039433 This permits copying and redistribution of the article or any adaptations for non-commercial purposes

Protein-free division of giant unilamellar vesicles controlled by enzymatic activity

Cell division is one of the hallmarks of life. Success in the bottom-up assembly of synthetic cells will, no doubt, depend on strategies for the controlled autonomous division of protocellular compartments. Here, we describe the protein-free division of giant unilamellar lipid vesicles (GUVs) based on the combination of two physical principles – phase separation and osmosis. We visualize the division process with confocal fluorescence microscopy and derive a conceptual model based on the vesicle geometry. The model successfully predicts the shape transformations over time as well as the time point of the final pinching of the daughter vesicles. Remarkably, we show that two fundamentally distinct yet highly abundant processes – water evaporation and metabolic activity – can both regulate the autonomous division of GUVs. Our work may hint towards mechanisms that governed the division of protocells and adds to the strategic toolbox of bottom-up synthetic biology with its vision of bringing matter to life

Light-triggered cargo loading and division of DNA-containing giant unilamellar lipid vesicles

A minimal synthetic cell should contain a substrate for information storage and have the capability to divide. Notable efforts were made to assemble functional synthetic cells from the bottom up, however often lacking the capability to reproduce. Here, we develop a mechanism to fully control reversible cargo loading and division of DNA-containing giant unilamellar vesicles (GUVs) with light. We make use of the photosensitizer Chlorin e6 (Ce6) which self-assembles into lipid bilayers and leads to local lipid peroxidation upon illumination. On the time scale of minutes, illumination induces the formation of transient pores, which we exploit for cargo encapsulation or controlled release. In combination with osmosis, complete division of two daughter GUVs can be triggered within seconds of illumination due to a spontaneous curvature increase. We ultimately demonstrate the division of a selected DNA-containing GUV with full spatiotemporal control—proving the relevance of the division mechanism for bottom-up synthetic biology

Printing and erasing of DNA-based photoresists inside synthetic cells

In the pursuit of producing functioning synthetic cells from the bottom-up, DNA nanotechnology has proven to be a powerful tool. However, the crowded yet highly organized arrangement in living cells, bridging from the nano- to the micron-scale, remains challenging to recreate with DNA-based architectures. Here, laser microprinting is established to print and erase shape-controlled DNA hydrogels inside the confinement of water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). The DNA-based photoresist consists of a photocleavable inactive DNA linker, which interconnects Y-shaped DNA motifs when activated by local irradiation with a 405 nm laser. An alternative linker design allows to erase custom features from a preformed DNA hydrogel with feature sizes down to 1.38 μm. The present work demonstrates that the DNA hydrogels can serve as an internal support to stabilize non-spherical GUV shapes. Overall, DNA-based photoresists for laser printing in confinement allow to build up architectures on the interior of synthetic cells with light, which diversifies the toolbox of bottom-up synthetic biology

Functionalization of cellular membranes with DNA nanotechnology

Due to its versatility and programmability, DNA nanotechnology has greatly expanded the experimental toolbox for biomedical research. Recent advances allow reliable and efficient functionalization of cellular plasma membranes with a variety of synthetic DNA constructs, ranging from single strands to complex 3D DNA origami. The scope for applications, which probe biophysical parameters or equip cells with novel functions, is rapidly increasing. These applications extend from programmed cellular connectivity and tissue engineering to molecular force measurements, controlled receptor–ligand interactions, membrane-anchored biosensors, and artificial transmembrane structures. Here, we give guidance on different strategies to functionalize cellular membranes with DNA nanotechnology and summarize current trends employing membrane-anchored DNA as a tool in biophysics, cell biology, and synthetic biology

Mastering complexity: towards bottom-up construction of multifunctional eukaryotic synthetic cells

Bottom-up synthetic biology thrives in reverse-engineering a particular biological function using a minimal set of molecular components, like purified proteins. Recently, precision technologies, like microfluidics, have been used to recombine functional modules towards multifunctional synthetic cells. Synthetic biology can capitalize on a variety of pre-existing on-chip functions, which greatly increases the scope for complexity in the field. Advances in DNA nanotechnology gave rise to a diverse range of fully synthetic functional modules, like DNA-based ion channels or motors, which can replace some protein-based parts. Noteworthy progress has been made in achieving large and stable compartments, organelle-like multicompartment systems, and sophisticated cytoskeletal structures. With the ultimate aim to construct a living cell, bottom-up synthetic biology strives to reconstitute cellular phenomena in vitro – disentangled from the complex environment of a cell. Recent work towards this ambitious goal has provided new insights into the mechanisms governing life. With the fast-growing library of functional modules for synthetic cells, their classification and integration become increasingly important. We discuss strategies to reverse-engineer and recombine functional parts for synthetic eukaryotes, mimicking the characteristics of nature’s own prototype. Particularly, we focus on large outer compartments, complex endomembrane systems with organelles, and versatile cytoskeletons as hallmarks of eukaryotic life. Moreover, we identify microfluidics and DNA nanotechnology as two technologies that can integrate these functional modules into sophisticated multifunctional synthetic cells

Aus dem Baukasten der molekularen Ingenieure. Auf dem Weg zur synthetischen Zelle

Die synthetische Biologie will lebensähnliche Materialsysteme auf der Basis ingenieurwissenschaftlicher Prinzipien kreieren. Was verwegen klingt, ist dank neuer Techniken in den Bereich des Möglichen gerückt

Functional DNA-based cytoskeletons for synthetic cells

The cytoskeleton is an essential component of a cell. It controls the cell shape, establishes the internal organization, and performs vital biological functions. Building synthetic cytoskeletons that mimic key features of their natural counterparts delineates a crucial step towards synthetic cells assembled from the bottom up. To this end, DNA nanotechnology represents one of the most promising routes, given the inherent sequence specificity, addressability and programmability of DNA. Here we demonstrate functional DNA-based cytoskeletons operating in microfluidic cell-sized compartments. The synthetic cytoskeletons consist of DNA tiles self-assembled into filament networks. These filaments can be rationally designed and controlled to imitate features of natural cytoskeletons, including reversible assembly and ATP-triggered polymerization, and we also explore their potential for guided vesicle transport in cell-sized confinement. Also, they possess engineerable characteristics, including assembly and disassembly powered by DNA hybridization or aptamer–target interactions and autonomous transport of gold nanoparticles. This work underpins DNA nanotechnology as a key player in building synthetic cells