Fast, multicolour optical sectioning over extended fields of view with patterned illumination and machine learning.

Structured illumination can reject out-of-focus signal from a sample, enabling high-speed and high-contrast imaging over large areas with widefield detection optics. However, this optical sectioning technique is currently limited by image reconstruction artefacts and poor performance at low signal-to-noise ratios. We combine multicolour interferometric pattern generation with machine learning to achieve high-contrast, real-time reconstruction of image data that is robust to background noise and sample motion. We validate the method in silico and demonstrate imaging of diverse specimens, from fixed and live biological samples to synthetic biosystems, reconstructing data live at 11 Hz across a 44 × 44µm2 field of view, and demonstrate image acquisition speeds exceeding 154 Hz.

The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that exist on a clinico-pathogenetic spectrum, designated ALS/FTD. The most common genetic cause of ALS/FTD is expansion of the intronic hexanucleotide repeat (GGGGCC)n in C9orf72. Here, we investigate the formation of nucleic acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of ALS/FTD. We observe significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associate to the formation of multimolecular G-quadruplexes (mG4s) by using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions leads to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validate the biological relevance of our findings by detecting an increased prevalence of G4-structures in C9orf72 mutant human motor neurons when compared to healthy motor neurons by staining with a G4-selective fluorescent probe, revealing signal in putative condensates. Our findings strongly suggest that RNA G-rich repetitive sequences can form protein-free condensates sustained by multimolecular G-quadruplexes, highlighting their potential relevance as therapeutic targets for C9orf72 mutation-related ALS/FTD.

DNA-Origami Line-Actants Control Domain Organization and Fission in Synthetic Membranes.

Cells can precisely program the shape and lateral organization of their membranes using protein machinery. Aiming to replicate a comparable degree of control, here we introduce DNA-origami line-actants (DOLAs) as synthetic analogues of membrane-sculpting proteins. DOLAs are designed to selectively accumulate at the line-interface between coexisting domains in phase-separated lipid membranes, modulating the tendency of the domains to coalesce. With experiments and coarse-grained simulations, we demonstrate that DOLAs can reversibly stabilize two-dimensional analogues of Pickering emulsions on synthetic giant liposomes, enabling dynamic programming of membrane lateral organization. The control afforded over membrane structure by DOLAs extends to three-dimensional morphology, as exemplified by a proof-of-concept synthetic pathway leading to vesicle fission. With DOLAs we lay the foundations for mimicking, in synthetic systems, some of the critical membrane-hosted functionalities of biological cells, including signaling, trafficking, sensing, and division.

Interplay of the mechanical and structural properties of DNA nanostructures determines their electrostatic interactions with lipid membranes.

Nucleic acids and lipids function in close proximity in biological processes, as well as in nanoengineered constructs for therapeutic applications. As both molecules carry a rich charge profile, and frequently coexist in complex ionic solutions, the electrostatics surely play a pivotal role in interactions between them. Here we discuss how each component of a DNA/ion/lipid system determines its electrostatic attachment. We examine membrane binding of a library of DNA molecules varying from nanoengineered DNA origami through plasmids to short DNA domains, demonstrating the interplay between the molecular structure of the nucleic acid and the phase of lipid bilayers. Furthermore, the magnitude of DNA/lipid interactions is tuned by varying the concentration of magnesium ions in the physiologically relevant range. Notably, we observe that the structural and mechanical properties of DNA are critical in determining its attachment to lipid bilayers and demonstrate that binding is correlated positively with the size, and negatively with the flexibility of the nucleic acid. The findings are utilized in a proof-of-concept comparison of membrane interactions of two DNA origami designs - potential nanotherapeutic platforms - showing how the results can have a direct impact on the choice of DNA geometry for biotechnological applications.

DNA-Based Optical Quantification of Ion Transport across Giant Vesicles.

Accurate measurements of ion permeability through cellular membranes remains challenging due to the lack of suitable ion-selective probes. Here we use giant unilamellar vesicles (GUVs) as membrane models for the direct visualization of mass translocation at the single-vesicle level. Ion transport is indicated with a fluorescently adjustable DNA-based sensor that accurately detects sub-millimolar variations in K+ concentration. In combination with microfluidics, we employed our DNA-based K+ sensor for extraction of the permeation coefficient of potassium ions. We measured K+ permeability coefficients at least 1 order of magnitude larger than previously reported values from bulk experiments and show that permeation rates across the lipid bilayer increase in the presence of octanol. In addition, an analysis of the K+ flux in different concentration gradients allows us to estimate the complementary H+ flux that dissipates the charge imbalance across the GUV membrane. Subsequently, we show that our sensor can quantify the K+ transport across prototypical cation-selective ion channels, gramicidin A and OmpF, revealing their relative H+/K+ selectivity. Our results show that gramicidin A is much more selective to protons than OmpF with a H+/K+ permeability ratio of ∼104.

Amphiphilic DNA nanostructures for bottom-up synthetic biology.

DNA nanotechnology enables the construction of sophisticated biomimetic nanomachines that are increasingly central to the growing efforts of creating complex cell-like entities from the bottom-up. DNA nanostructures have been proposed as both structural and functional elements of these artificial cells, and in many instances are decorated with hydrophobic moieties to enable interfacing with synthetic lipid bilayers or regulating bulk self-organisation. In this feature article we review recent efforts to design biomimetic membrane-anchored DNA nanostructures capable of imparting complex functionalities to cell-like objects, such as regulated adhesion, tissue formation, communication and transport. We then discuss the ability of hydrophobic modifications to enable the self-assembly of DNA-based nanostructured frameworks with prescribed morphology and functionality, and explore the relevance of these novel materials for artificial cell science and beyond. Finally, we comment on the yet mostly unexpressed potential of amphiphilic DNA-nanotechnology as a complete toolbox for bottom-up synthetic biology - a figurative and literal scaffold upon which the next generation of synthetic cells could be built.

Thermally Driven Membrane Phase Transitions Enable Content Reshuffling in Primitive Cells.

Self-assembling single-chain amphiphiles available in the prebiotic environment likely played a fundamental role in the advent of primitive cell cycles. However, the instability of prebiotic fatty acid-based membranes to temperature and pH seems to suggest that primitive cells could only host prebiotically relevant processes in a narrow range of nonfluctuating environmental conditions. Here we propose that membrane phase transitions, driven by environmental fluctuations, enabled the generation of daughter protocells with reshuffled content. A reversible membrane-to-oil phase transition accounts for the dissolution of fatty acid-based vesicles at high temperatures and the concomitant release of protocellular content. At low temperatures, fatty acid bilayers reassemble and encapsulate reshuffled material in a new cohort of protocells. Notably, we find that our disassembly/reassembly cycle drives the emergence of functional RNA-containing primitive cells from parent nonfunctional compartments. Thus, by exploiting the intrinsic instability of prebiotic fatty acid vesicles, our results point at an environmentally driven tunable prebiotic process, which supports the release and reshuffling of oligonucleotides and membrane components, potentially leading to a new generation of protocells with superior traits. In the absence of protocellular transport machinery, the environmentally driven disassembly/assembly cycle proposed herein would have plausibly supported protocellular content reshuffling transmitted to primitive cell progeny, hinting at a potential mechanism important to initiate Darwinian evolution of early life forms.

Responsive core-shell DNA particles trigger lipid-membrane disruption and bacteria entrapment.

Biology has evolved a variety of agents capable of permeabilizing and disrupting lipid membranes, from amyloid aggregates, to antimicrobial peptides, to venom compounds. While often associated with disease or toxicity, these agents are also central to many biosensing and therapeutic technologies. Here, we introduce a class of synthetic, DNA-based particles capable of disrupting lipid membranes. The particles have finely programmable size, and self-assemble from all-DNA and cholesterol-DNA nanostructures, the latter forming a membrane-adhesive core and the former a protective hydrophilic corona. We show that the corona can be selectively displaced with a molecular cue, exposing the 'sticky' core. Unprotected particles adhere to synthetic lipid vesicles, which in turn enhances membrane permeability and leads to vesicle collapse. Furthermore, particle-particle coalescence leads to the formation of gel-like DNA aggregates that envelop surviving vesicles. This response is reminiscent of pathogen immobilisation through immune cells secretion of DNA networks, as we demonstrate by trapping E. coli bacteria.

Cations Regulate Membrane Attachment and Functionality of DNA Nanostructures.

The interplay between nucleic acids and lipids underpins several key processes in molecular biology, synthetic biotechnology, vaccine technology, and nanomedicine. These interactions are often electrostatic in nature, and much of their rich phenomenology remains unexplored in view of the chemical diversity of lipids, the heterogeneity of their phases, and the broad range of relevant solvent conditions. Here we unravel the electrostatic interactions between zwitterionic lipid membranes and DNA nanostructures in the presence of physiologically relevant cations, with the purpose of identifying new routes to program DNA-lipid complexation and membrane-active nanodevices. We demonstrate that this interplay is influenced by both the phase of the lipid membranes and the valency of the ions and observe divalent cation bridging between nucleic acids and gel-phase bilayers. Furthermore, even in the presence of hydrophobic modifications on the DNA, we find that cations are still required to enable DNA adhesion to liquid-phase membranes. We show that the latter mechanism can be exploited to control the degree of attachment of cholesterol-modified DNA nanostructures by modifying their overall hydrophobicity and charge. Besides their biological relevance, the interaction mechanisms we explored hold great practical potential in the design of biomimetic nanodevices, as we show by constructing an ion-regulated DNA-based synthetic enzyme.

A Modular, Dynamic, DNA-Based Platform for Regulating Cargo Distribution and Transport between Lipid Domains.

Cell membranes regulate the distribution of biological machinery between phase-separated lipid domains to facilitate key processes including signaling and transport, which are among the life-like functionalities that bottom-up synthetic biology aims to replicate in artificial-cellular systems. Here, we introduce a modular approach to program partitioning of amphiphilic DNA nanostructures in coexisting lipid domains. Exploiting the tendency of different hydrophobic "anchors" to enrich different phases, we modulate the lateral distribution of our devices by rationally combining hydrophobes and by changing nanostructure size and topology. We demonstrate the functionality of our strategy with a bioinspired DNA architecture, which dynamically undergoes ligand-induced reconfiguration to mediate cargo transport between domains via lateral redistribution. Our findings pave the way to next-generation biomimetic platforms for sensing, transduction, and communication in synthetic cellular systems.