62 results found
Walczak M, Mancini L, Xu J, et al., 2023, A Synthetic Signaling Network Imitating the Action of Immune Cells in Response to Bacterial Metabolism., Adv Mater, Vol: 35
State-of-the-art bottom-up synthetic biology allows to replicate many basic biological functions in artificial-cell-like devices. To mimic more complex behaviors, however, artificial cells would need to perform many of these functions in a synergistic and coordinated fashion, which remains elusive. Here, a sophisticated biological response is considered, namely the capture and deactivation of pathogens by neutrophil immune cells, through the process of netosis. A consortium consisting of two synthetic agents is designed-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 immobilization and exposure to antibiotics. The 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.
Rubio-Sánchez R, Mognetti BM, Cicuta P, et al., 2023, DNA-Origami Line-Actants Control Domain Organization and Fission in Synthetic Membranes., J Am Chem Soc, Vol: 145, Pages: 11265-11275
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.
Garcia Hernandez NV, Buccelli S, Laffranchi M, et al., 2023, Mixed Reality-based Exergames for Upper Limb Robotic Rehabilitation, HRI '23: ACM/IEEE International Conference on Human-Robot Interaction, Publisher: ACM
Walczak M, Brady RA, Leathers A, et al., 2023, Influence of hydrophobic moieties on the crystallization of amphiphilic DNA nanostructures, JOURNAL OF CHEMICAL PHYSICS, Vol: 158, ISSN: 0021-9606
Walczak M, Mancini L, Xu J, et al., 2023, A synthetic signalling network imitating the action of immune cells in response to bacterial metabolism
<jats:p>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,<jats:italic>artificial cells</jats:italic>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.</jats:p>
Raguseo F, Huyghebaert A, Li J, et al., 2023, The ALS/FTD-related C9orf72 hexanucleotide repeat expansion forms RNA condensates through multimolecular G-quadruplexes
<jats:title>Abstract</jats:title><jats:p>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)<jats:italic><jats:sub>n</jats:sub></jats:italic>in<jats:italic>C9orf72</jats:italic>. 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)<jats:italic><jats:sub>n</jats:sub></jats:italic>, 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<jats:italic>C9orf72</jats:italic>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<jats:italic>C9orf72</jats:italic>mutation related ALS and FTD.</jats:p><jats:p><jats:fig id="ufig1" position="float" orientation="portrait" fig-type="figure"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="526399v3_ufig1" position="float" orientation="portrait" /></jats:fig></jats:p>
Morzy D, Tekin C, Caroprese V, et al., 2023, Interplay of the mechanical and structural properties of DNA nanostructures determines their electrostatic interactions with lipid membranes, NANOSCALE, Vol: 15, Pages: 2849-2859, ISSN: 2040-3364
Rubio-Sánchez R, Mognetti BM, Cicuta P, et al., 2023, DNA-origami line-actants control domain organisation and fission in synthetic membranes
<jats:title>Abstract</jats:title><jats:p>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.</jats:p>
Takamori S, Cicuta P, Takeuchi S, et al., 2022, DNA-assisted selective electrofusion (DASE) of Escherichia coli and giant lipid vesicles, Nanoscale, Vol: 14, Pages: 14255-14267, ISSN: 2040-3364
Synthetic biology and cellular engineering require chemical and physical alterations, which are typically achieved by fusing target cells with each other or with payload-carrying vectors. On one hand, electrofusion can efficiently induce the merging of biological cells and/or synthetic analogues via the application of intense DC pulses, but it lacks selectivity and often leads to uncontrolled fusion. On the other hand, synthetic DNA-based constructs, inspired by natural fusogenic proteins, have been shown to induce a selective fusion between membranes, albeit with low efficiency. Here we introduce DNA-assisted selective electrofusion (DASE) which relies on membrane-anchored DNA constructs to bring together the objects one seeks to merge, and applying an electric impulse to trigger their fusion. The DASE process combines the efficiency of standard electrofusion and the selectivity of fusogenic nanostructures, as we demonstrate by inducing and characterizing the fusion of spheroplasts derived from Escherichia coli bacteria with cargo-carrying giant lipid vesicles.
Fletcher M, Zhu J, Rubio-Sanchez R, et al., 2022, DNA-Based Optical Quantification of Ion Transport across Giant Vesicles, ACS NANO, Vol: 16, Pages: 17128-17138, ISSN: 1936-0851
Leathers A, Walczak M, Brady R, et al., 2022, Reaction-diffusion patterning of DNA-based artificial cells, Journal of the American Chemical Society, Vol: 144, Pages: 17468-17476, ISSN: 0002-7863
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 forconstructing membrane-less artificial cells from the self-assembly of synthetic DNA nanostructures, in which internal domains can be estab-lished 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 RNAaptamers, which then accumulate in a surrounding storage shell, thus demonstrating spatial segregation of functionalities reminiscent of that observed in biological cells.
Paez Perez M, Russell A, Cicuta P, et al., 2022, Modulating membrane fusion through the design of fusogenic DNA circuits and bilayer composition, Soft Matter, Vol: 18, Pages: 7035-7044, ISSN: 1744-683X
Membrane fusion is a ubiquitous phenomenon linked to many biological processes, and representsa crucial step in liposome-based drug delivery strategies. The ability to control, ever more precisely,membrane fusion pathways would thus be highly valuable for next generation nano-medical solutions and, more generally, the design of advanced biomimetic systems such as synthetic cells. Inthis article, we present fusogenic nanostructures constructed from synthetic DNA which, differentfrom previous solutions, unlock routes for modulating the rate of fusion and making it conditionalto the presence of soluble DNA molecules, thus demonstrating how membrane fusion can be controlled through simple DNA-based molecular circuits. We then systematically explore the relationshipbetween lipid-membrane composition, its biophysical properties, and measured fusion efficiency, linking our observations to the stability of transition states in the fusion pathway. Finally, we observethat specific lipid compositions lead to the emergence of complex bilayer architectures in the fusion products, such as nested morphologies, which are accompanied by alterations in biophysicalbehaviour. Our findings provide multiple, orthogonal strategies to program lipid-membrane fusion,which leverage the design of either the fusogenic DNA constructs or the physico/chemical propertiesof the membranes, and could thus be valuable in applications where some design parameters areconstrained by other factors such as material cost and biocompatibility, as it is often the case inbiotechnological applications.
Kaufhold WT, Pfeifer W, Castro CE, et al., 2022, Probing the mechanical properties of DNA nanostructures with metadynamics, ACS Nano, Vol: 16, Pages: 8784-8797, ISSN: 1936-0851
Molecular dynamics simulations are often used to provide feedback in the design workflow of DNA nanostructures. However, even with coarse-grained models, the convergence of distributions from unbiased simulation is slow, limiting applications to equilibrium structural properties. Given the increasing interest in dynamic, reconfigurable, and deformable devices, methods that enable efficient quantification of large ranges of motion, conformational transitions, and mechanical deformation are critically needed. Metadynamics is an automated biasing technique that enables the rapid acquisition of molecular conformational distributions by flattening free energy landscapes. Here we leveraged this approach to sample the free energy landscapes of DNA nanostructures whose unbiased dynamics are nonergodic, including bistable Holliday junctions and part of a bistable DNA origami structure. Taking a DNA origami-compliant joint as a case study, we further demonstrate that metadynamics can predict the mechanical response of a full DNA origami device to an applied force, showing good agreement with experiments. Our results exemplify the efficient computation of free energy landscapes and force response in DNA nanodevices, which could be applied for rapid feedback in iterative design workflows and generally facilitate the integration of simulation and experiments. Metadynamics will be particularly useful to guide the design of dynamic devices for nanorobotics, biosensing, or nanomanufacturing applications.
Fabrini G, Minard A, Brady R, et al., 2022, Cation-responsive and photocleavable hydrogels from non-canonical amphiphilic DNA nanostructures, Nano Letters: a journal dedicated to nanoscience and nanotechnology, Vol: 22, Pages: 602-611, ISSN: 1530-6984
Thanks to its biocompatibility, versatility and programmable interactions, DNA has been proposed as a building block for functional, stimuli-responsive frameworks with applications in biosensing, tissue engineering and drug delivery. Of particular importance for in vivo applications is the possibility of making such nano-materials responsive to physiological stimuli. Here we demonstrate how combining non-canonical DNA G-quadruplex (G4) structures with amphiphilic DNA constructs yields nanos-tructures, which we termed “Quad-Stars”, capable of assembling into responsive hydrogel particles via a straightforward, enzyme-free, one-pot reaction. The embedded G4 structures allow one to trigger and control the assembly/disassembly in a reversible fashion by adding or removing K+ ions. Furthermore, the hydrogel aggregates can be photo disassembled upon near-UV irradiation in the presence of a porphyrin photosensitiser. The combinedreversibility of assembly, responsiveness and cargo-loading capabilities of the hydrophobic moieties make Quad-Stars a promising candidate for biosensors and responsive drug delivery carriers.
Rubio-Sanchez R, Fabrini G, Cicuta P, et al., 2021, Amphiphilic DNA nanostructures for bottom-up synthetic biology, Chemical Communications, Vol: 57, Pages: 12725-12740, ISSN: 1359-7345
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.
Rubio-Sanchez R, O'Flaherty DK, Wang A, et al., 2021, Thermally driven membrane phase transitions enable content reshuffling in primitive cells, Journal of the American Chemical Society, Vol: 143, Pages: 16589-16598, ISSN: 0002-7863
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.
Walczak M, Brady RA, Mancini L, et al., 2021, Responsive core-shell DNA particles trigger lipid-membrane disruption and bacteria entrapment, Nature Communications, Vol: 12, Pages: 1-11, ISSN: 2041-1723
Biology has evolved a variety of agents capable of permeabilising and disrupting lipid membranes, from amyloid aggregates, to antimicrobial peptides, to venom compounds. While often associatedwith disease or toxicity, these agents are also central to many biosensing and therapeutic tech nologies. 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 molecu19 lar 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.
Morzy D, Rubio-Sanchez R, Joshi H, et al., 2021, Cations regulate membrane attachment and functionality of DNA nanostructures, Journal of the American Chemical Society, Vol: 143, Pages: 7358-7367, ISSN: 0002-7863
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.
Rubio R, Eizagirre Barker S, Walczak M, et al., 2021, A modular, dynamic, DNA-based platform for regulating cargo distribution and transport between lipid domains, Nano Letters, Vol: 21, Pages: 2800-2808, ISSN: 1530-6984
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.
Rubio-Sánchez R, Barker SE, Walczak M, et al., 2021, 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.
Clowsley AH, Kaufhold WT, Lutz T, et al., 2021, Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy, Nature Communications, Vol: 12, Pages: 1-10, ISSN: 2041-1723
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.
Lanfranco R, Jana PK, Bruylants G, et al., 2020, Adaptable DNA interactions regulate surface triggered self assembly, Nanoscale, Vol: 12, Pages: 18616-18620, ISSN: 2040-3364
DNA-mediated multivalent interactions between colloidal particles have been extensively applied for their ability to program bulk phase behaviour and dynamic processes. Exploiting the competition between different types of DNA–DNA bonds, here we experimentally demonstrate the selective triggering of colloidal self-assembly in the presence of a functionalised surface, which induces changes in particle–particle interactions. Besides its relevance to the manufacturing of layered materials with controlled thickness, the intrinsic signal-amplification features of the proposed interaction scheme make it valuable for biosensing applications.
Clowsley AH, Kaufhold WT, Lutz T, et al., 2020, Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy, Journal of the American Chemical Society, Vol: 142, Pages: 12069-12078, ISSN: 0002-7863
Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution, which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present proximity-dependent PAINT (PD-PAINT), a method for subdiffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA constructs anchored to the target species. Labeled protein pairs are then imaged with high-contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution, and the ability to image quantitatively.
Mognetti BM, Cicuta P, Di Michele L, 2019, Programmable interactions with biomimetic DNA linkers at fluid membranes and interfaces., Reports on Progress in Physics, Vol: 82, Pages: 1-34, ISSN: 0034-4885
At the heart of the structured architecture and complex dynamics of biological&#13; systems are speciﬁc and timely interactions operated by biomolecules. In many&#13; instances, biomolecular agents are spatially conﬁned to ﬂexible lipid membranes where,&#13; among other functions, they control cell adhesion, motility and tissue formation.&#13; Besides being central to several biological processes, multivalent interactions mediated&#13; by reactive linkers conﬁned to deformable substrates underpin the design of synthetic-&#13; biological platforms and advanced biomimetic materials. Here we review recent&#13; advances on the experimental study and theoretical modelling of a heterogeneous&#13; class of biomimetic systems in which synthetic linkers mediate multivalent interactions&#13; between ﬂuid and deformable colloidal units, including lipid vesicles and emulsion&#13; droplets. Linkers are often prepared from synthetic DNA nanostructures, enabling&#13; full programmability of the thermodynamic and kinetic properties of their mutual&#13; interactions. The coupling of the statistical eﬀects of multivalent interactions with&#13; substrate ﬂuidity and deformability gives rise to a rich emerging phenomenology that,&#13; in the context of self-assembled soft materials, has been shown to produce exotic phase&#13; behaviour, stimuli-responsiveness, and kinetic programmability of the self-assembly&#13; process. Applications to (synthetic) biology will also be reviewed.
del Barrio J, Liu J, Brady RA, et al., 2019, Emerging Two-Dimensional Crystallization of Cucurbituril Complexes: From Supramolecular Polymers to Nanofibers, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 141, Pages: 14021-14025, ISSN: 0002-7863
Kaufhold WT, Brady RA, Tuffnell JM, et al., 2019, Membrane Scaffolds Enhance the Responsiveness and Stability of DNA-Based Sensing Circuits, BIOCONJUGATE CHEMISTRY, Vol: 30, Pages: 1850-1859, ISSN: 1043-1802
Talbot EL, Kotar J, Di Michele L, et al., 2019, Directed tubule growth from giant unilamellar vesicles in a thermal gradient, SOFT MATTER, Vol: 15, Pages: 1676-1683, ISSN: 1744-683X
Lanfranco R, Jana PK, Tunesi L, et al., 2019, Kinetics of Nanoparticle-Membrane Adhesion Mediated by Multivalent Interactions, LANGMUIR, Vol: 35, Pages: 2002-2012, ISSN: 0743-7463
Brady RA, Kaufhold WT, Brooks NJ, et al., 2019, Flexibility defines structure in crystals of amphiphilic DNA nanostars, JOURNAL OF PHYSICS-CONDENSED MATTER, Vol: 31, ISSN: 0953-8984
Lutz T, Clowsley AH, Lin R, et al., 2018, Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT, Nano Research, Vol: 11, Pages: 6141-6154, ISSN: 1998-0124
The optical super-resolution technique DNA-PAINT (Point Accumulation Imaging in Nanoscale Topography) provides a flexible way to achieve imaging of nanoscale structures at ∼10-nanometer resolution. In DNA-PAINT, fluorescently labeled DNA “imager” strands bind transiently and with high specificity to complementary target “docking” strands anchored to the structure of interest. The localization of single binding events enables the assembly of a super-resolution image, and this approach effectively circumvents photobleaching. The solution exchange of imager strands is the basis of Exchange-PAINT, which enables multiplexed imaging that avoids chromatic aberrations. Fluid exchange during imaging typically requires specialized chambers or washes, which can disturb the sample. Additionally, diffusional washout of imager strands is slow in thick samples such as biological tissue slices. Here, we introduce Quencher-Exchange-PAINT—a new approach to Exchange-PAINT in regular open-top imaging chambers—which overcomes the comparatively slow imager strand switching via diffusional imager washout. Quencher-Exchange-PAINT uses “quencher” strands, i.e., oligonucleotides that prevent the imager from binding to the targets, to rapidly reduce unwanted single-stranded imager concentrations to negligible levels, decoupled from the absolute imager concentration. The quencher strands contain an effective dye quencher that reduces the fluorescence of quenched imager strands to negligible levels. We characterized Quencher-Exchange-PAINT when applied to synthetic, cellular, and thick tissue samples. Quencher-Exchange-PAINT opens the way for efficient multiplexed imaging of complex nanostructures, e.g., in thick tissues, without the need for washing steps. [Figure not available: see fulltext.].
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