Biology subjects

Di Michele, L.

Publications and source records attributed to Di Michele, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Characterizing MINFLUX imaging performance with DNA origami

MINFLUX, a second-generation super-resolution technique, can localize fluorescent markers approaching single-nanometer precision in three dimensions. Similar to previous super-resolution methodologies, the extended duration of acquisitions can result in drift that needs accurate correction to enable proper analysis and interpretation of the collected data. Here, we use DNA origami, housing sites of known spatial distribution fitted with repeat-domain docking strands for DNA-PAINT imaging to characterize imaging performance over extended duration MINFLUX acquisitions (6-20 h). Repeat-domain docking strands overcome site-loss and reveal residual drift in prolonged MINFLUX 3D acquisitions that we correct with an algorithm exploiting time-correlated shifts of localizations around identified DNA origami sites. Following correction of residual drift the site precision, i.e. the scatter of localizations around sites, is [~]2 nm in all directions. Comparison of site precision from extended repeat-domain docking strands with site precision from standard short 8-10 nucleotide docking strands exhibits no detectable loss of site precision. By adding DNA origami structures to mounted biological samples we apply our approach to the imaging of the cardiac ryanodine receptor 2 in cryosectioned heart tissue. The data suggests that for these protein targets single domain markers with repeat domain docking strands may be directly used for residual drift correction, simplifying sample preparation and acquisition protocols.

biophysics↗

bioRxiv2025

Organization and triggered release of liposomes with DNA-based synthetic condensates

Cells use a combination of membrane-bound and membrane-less compartments to dynamically orchestrate internal biochemical processes and sustain intracellular communication. Recapitulating the hierarchical integration and interplay between these physically and chemically diverse structures is required to enhance the functionalities of synthetic cells and other advanced biomimetic systems. Here, we describe the use of synthetic DNA condensates to selectively uptake and spatially organize lipid vesicles, interacting with the condensates thanks to cholesterol-DNA anchors. By modulating anchor density, the liposomes can be programmably localized on the surface or interior of the condensates, while base-pairing selectivity can be leveraged to target individual internal domains in multi-phasic condensates. The embedded liposomes can be released by adding a nucleic acid trigger and captured by a second condensate population, thus imitating extracellular vesicles in their ability to support long-range cellular communication. This modular platform demonstrates the potential of DNA-based condensates to program the spatial distribution of membranous subcompartments and to support dynamic cargo-handling capabilities. These features are valuable for engineering cell mimics, microreactors, and delivery systems.

synthetic biology↗

bioRxiv2025

Stimuli-driven cyclical content exchange in a composite synthetic cell

Engineering sophisticated behaviours in synthetic cells lacking complex biomolecular machinery remains a central challenge in synthetic biology. Here, we introduce a protein-free approach for dynamic content modulation in liposome-based synthetic cells using an internal gelation strategy. By crosslinking a polymer hydrogel within the lumen of giant vesicles and tethering it to the inner membrane leaflet, we create a composite architecture that enables controlled and reversible membrane permeabilisation via osmotic swelling and shrinking, facilitating externally gated material exchange without reconstituted protein pores or electroporation. Simultaneously, the hydrogel matrix affords control over membrane fluidity and the diffusion of cytoplasmic clients. We deploy the transport-regulation platform to construct a synthetic-cell bioreactor whereby reversible membrane permeabilisation enables content supplementation and fuels a biocatalytic reaction. The composite gel-GUV chassis provides an adaptive, robust and expandable solution for engineering increasingly modular and functional synthetic cellular systems. These findings may echo how primordial cells harnessed environmental fluctuations for content exchange through chemically distinct pathways.

synthetic biology↗

bioRxiv2025

Expression of nano-engineered RNA organelles in bacteria

Designing synthetic biomolecular condensates, or membrane-less organelles, offers insights on the functions of their natural counterparts, and is equally valuable for cellular and metabolic engineering. Choosing E. coli for its biotechnological relevance, we deploy RNA nanotechnology to design and express non-natural membrane-less organelles in vivo. The designer condensates assemble co-transcriptionally from branched RNA motifs interacting via base-pairing. Exploiting binding selectivity we express orthogonal, non-mixing condensates, and by embedding a protein-binding aptamer we achieve selective protein recruitment. Condensates can be made to dissolve and re-assemble upon thermal cycling, thereby reversibly releasing and re-capturing protein clients. The synthetic organelles are expressed robustly across the cell population and remain stable despite enzymatic RNA processing. Compared with existing solutions based on peptide building blocks or repetitive RNA sequences, these nanostructured RNA motifs enable algorithmic control over interactions, affinity for clients, and condensate microstructure, opening new directions in synthetic biology and biotechnology.

synthetic biology↗

bioRxiv2025

Cation-controlled assembly, activity, and organisation of biomimetic DNA receptors in synthetic cell membranes

Biological cells use cations as signalling messengers to regulate a variety of responses. Linking cations to the functionality of synthetic membranes is thus crucial to engineering advanced biomimetic agents, such as synthetic cells. Here, we introduce bio-inspired DNA-based receptors that exploit non-canonical G-quadruplexes for cation-actuated structural and functional responses in synthetic lipid membranes. Membrane confinement grants cationdependent control over receptor assembly and, when supplemented with hemin co-factors, their peroxidase DNAzyme activity. Cationmediated control extends to receptor lateral distribution to localise DNA-based catalysis within phase-separated membrane domains of model synthetic cells, imitating the localisation of multimeric membrane complexes to signalling hubs in living cells. Our modular strategy paves the way for engineering from the bottom-up cation-responsive pathways for sensing, signalling, and communication in synthetic cellular systems.

biophysics↗

bioRxiv2025

Internal phase separation in synthetic DNA condensates

Biomolecular condensates regulate cellular biochemistry by organizing enzymes, substrates and metabolites, and often acquire partially de-mixed states whereby distinct internal domains play functional roles. Despite their physiological relevance, questions remain about the principles underpinning the emergence of multi-phase condensates. Here, we present a model system of synthetic DNA nanostructures able to form monophasic or biphasic condensates. Key condensate features, including the degree of interphase mixing and the relative size and spatial arrangement of domains, can be controlled by altering nanostructure stoichiometries. The modular nature of the system facilitates an intuitive understanding of phase behavior, and enables mapping of the experimental phenomenology onto a predictive Flory-Huggins model. The experimental and theoretical framework we introduce will help address open questions on multiphase condensation in biology and aid the design of functional biomolecular condensates in vitro, in synthetic cells, and in living cells.

biophysics↗

bioRxiv2024

Enzyme-Responsive DNA Condensates

Membrane-less compartments and organelles are widely acknowledged for their role in regulating cellular processes, and there is an urgent need to harness their full potential as both structural and functional elements of synthetic cells. Despite rapid progress, synthetically recapitulating the nonequilibrium, spatially distributed responses of natural membrane-less organelles remain elusive. Here we demonstrate that the activity of nucleic-acid cleaving enzymes can be localised within DNA-based membrane-less compartments by sequestering the respective DNA or RNA substrates. Reaction-diffusion processes lead to complex nonequilibrium patterns, dependent on enzyme concentration. By arresting similar dynamic patterns, we spatially organise different substrates in concentric sub-compartments, which can be then selectively addressed by different enzymes, demonstrating spatial distribution of enzymatic activity. Besides advancing our ability to engineer advanced biomimetic functions in synthetic membrane-less organelles, our results may facilitate the deployment of DNA-based condensates as microbioreactors or platforms for the detection and quantitation of enzymes and nucleic acids.

synthetic biology↗

bioRxiv2024

Reduced non-specific binding of super-resolution DNA-PAINT markers by Shielded DNA-PAINT labeling protocols

The DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events [~]5 fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific background.

biophysics↗

bioRxiv2023

Modular RNA motifs for orthogonal phase separated compartments

Recent discoveries in biology have highlighted the importance of protein and RNA-based condensates as an alternative to classical membrane-bound organelles for the task of compartmentalizing molecules and biochemical reactions. Here, we demonstrate the rational design of pure RNA condensates from star-shaped RNA motifs. We generate condensates using two different RNA nanostar architectures: multi-stranded nanostars whose binding interactions are programmed via single-stranded overhangs, and single-stranded nanostars whose interactions are programmed via kissing loops. Through rational design of the nanostar interaction sequences, we demonstrate that both architectures can produce orthogonal (distinct and immiscible) condensates, which can be individually tracked via fluorogenic aptamers. We also show that aptamers make it possible to recruit peptides and proteins to the condensates with high specificity. Successful cotranscriptional formation of condensates from single-stranded nanostars suggests that they may be genetically encoded and produced in living cells. We provide a library of orthogonal RNA condensates that can be modularly customized and offer a route toward creating systems of functional artificial organelles.

synthetic biology↗

bioRxiv2023

Co-transcriptional production of programmable RNA condensates and synthetic organelles

Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Here we introduce a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co-transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate guest molecules. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with controlled number, size, morphology and composition, and that display the ability to selectively capture proteins. The in situ expression of programmable RNA condensates could underpin spatial organisation of functionalities in both biological and synthetic cells.

synthetic biology↗

bioRxiv2023

Sculpting DNA-based synthetic cells through phase separation and phase-targeted activity

Synthetic cells, like their biological counterparts, require internal compartments with distinct chemical and physical properties where different functionalities can be localised. Inspired by membrane-less compartmentalisation in biological cells, here we demonstrate how micro-phase separation can be used to engineer heterogeneous cell-like architectures with programmable morphology and compartment-targeted activity. The synthetic cells selfassemble from amphiphilic DNA nanostructures, producing core-shell condensates due to size-induced de-mixing. Lipid deposition and phase-selective etching are then used to generate a porous pseudo-membrane, a cytoplasm analogue, and membrane-less organelles. The synthetic cells can sustain RNA synthesis via in vitro transcription, leading to cytoplasm and pseudo-membrane expansion caused by an accumulation of the transcript. Our approach exemplifies how architectural and functional complexity can emerge from a limited number of distinct building blocks, if molecular-scale programmability, emergent biophysical phenomena, and biochemical activity are coupled to mimic those observed in live cells.

biophysics↗

bioRxiv2023

A synthetic signalling network imitating the action of immune cells in response to bacterial metabolism

State-of-the-art bottom-up synthetic biology allows us to replicate many basic biological functions in artificial cell-like devices. To mimic more complex behaviours, however, artificial cells would need to perform many of these functions in a synergistic and coordinated fashion, which remains elusive. Here we considered a sophisticated biological response, namely the capture and deactivation of pathogens by neutrophil immune cells, through the process of netosis. We designed a consortium consisting of two synthetic agents - responsive DNA-based particles and antibiotic-loaded lipid vesicles - whose coordinated action mimics the sought immune-like response when triggered by bacterial metabolism. The artificial netosis-like response emerges from a series of interlinked sensing and communication pathways between the live and synthetic agents, and translates into both physical and chemical antimicrobial actions, namely bacteria immobilisation and exposure to antibiotics. Our results demonstrate how advanced life-like responses can be prescribed with a relatively small number of synthetic molecular components, and outlines a new strategy for artificial-cell-based antimicrobial solutions.

synthetic biology↗

bioRxiv2023

The C9Orf72 hexanucleotide repeat expansion transcript forms insoluble aggregates facilitated by multimolecular G-quadruplex structures

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 the expansion of the intronic hexanucleotide repeat (GGGGCC)nin C9orf72. Here, we investigated the formation of nucleic-acid secondary structures in these expansion repeats, and their role in generating condensates characteristic of the diseases. We observed significant aggregation of the hexanucleotide sequence (GGGGCC)n, which we associated to the formation of multimolecular G-quadruplexes (mG4s), using a range of biophysical techniques. Exposing the condensates to G4-unfolding conditions led to prompt disassembly, highlighting the key role of mG4-formation in the condensation process. We further validated the biological relevance of our findings by demonstrating the ability of a G4-selective fluorescent probe to penetrate C9orf72 mutant human motor neurons derived from ALS patients, which revealed clear fluorescent 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 and FTD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/526399v3_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@18c11e6org.highwire.dtl.DTLVardef@eb308eorg.highwire.dtl.DTLVardef@c0c870org.highwire.dtl.DTLVardef@a7555a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

DNA-origami line-actants control domain organisation and fission in synthetic membranes

Cells can precisely program the shape and lateral organisation 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 co-existing 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 stabilise two-dimensional analogues of Pickering emulsions on synthetic giant liposomes, enabling dynamic programming of membrane lateral organisation. 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 signalling, trafficking, sensing, and division.

biophysics↗

bioRxiv2022

Reaction-diffusion patterning of DNA-based artificial cells

Biological cells display complex internal architectures, with distinct micro environments that establish the chemical heterogeneity needed to sustain cellular functions. The continued efforts to create advanced cell mimics - artificial cells - demands strategies to construct similarly heterogeneous structures with localized functionalities. Here, we introduce a platform for constructing membrane-less artificial cells from the self-assembly of synthetic DNA nanostructures, in which internal domains can be established thanks to prescribed reaction-diffusion waves. The method, rationalized through numerical modeling, enables the formation of up to five distinct, concentric environments, in which functional moieties can be localized. As a proof-of-concept, we apply this platform to build DNA-based artificial cells in which a prototypical nucleus synthesizes fluorescent RNA aptamers, which then accumulate in a surrounding storage shell, thus demonstrating spatial segregation of functionalities reminiscent of that observed in biological cells.

synthetic biology↗

bioRxiv2021

Thermal oscillations enable reshuffling of genetic material in a primitive cell cycle

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 non-fluctuating environmental conditions. Here we propose a novel primitive cell cycle driven by environmental fluctuations, which enable 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 genetic content. At low temperatures, fatty acid bilayers reassemble and encapsulate reshuffled genetic 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 non-functional compartments. Thus, by exploiting the intrinsic instability of prebiotic fatty acid vesicles, our results point at an environmentally-driven tunable primitive cell cycle, which supports the release and reshuffle of protocellular genetic 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 supported genetic content reshuffling transmitted to primitive cell progeny, hinting at a potential mechanism important to initiate Darwinian evolution of early lifeforms.

biophysics↗

bioRxiv2021

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 signalling 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 co-existing 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 its topology. We demonstrate the functionality of our strategy with a bio-inspired DNA architecture, which dynamically undergoes ligand-induced reconfiguration to mediate cargo transport between domains via lateral re-distribution. Our findings pave the way to next-generation biomimetic platforms for sensing, transduction, and communication in synthetic cellular systems.

biophysics↗

bioRxiv2020

Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy

DNA-PAINT is a versatile optical super-resolution technique relying on the transient binding of fluorescent DNA imagers to target epitopes. Its performance in biological samples is often constrained by strong background signals and non-specific binding events, both exacerbated by high imager concentrations. Here we describe Repeat DNA-PAINT, a method that enables a substantial reduction in imager concentration, thus suppressing spurious signals. Additionally, Repeat DNA-PAINT reduces photoinduced target-site loss and can accelerate sampling, all without affecting spatial resolution.

molecular biology↗