Tissue-like environments shape functional interactions of HIV-1 with immature dendritic cells.

Immature dendritic cells (iDCs) migrate in microenvironments with distinct cell and extracellular matrix densities in vivo and contribute to HIV-1 dissemination and mounting of antiviral immune responses. Here, we find that, compared to standard 2D suspension cultures, 3D collagen as tissue-like environment alters iDC properties and their response to HIV-1 infection. iDCs adopt an elongated morphology with increased deformability in 3D collagen at unaltered activation, differentiation, cytokine secretion, or responsiveness to LPS. While 3D collagen reduces HIV-1 particle uptake by iDCs, fusion efficiency is increased to elevate productive infection rates due to elevated cell surface exposure of the HIV-1-binding receptor DC-SIGN. In contrast, 3D collagen reduces HIV transfer to CD4 T cells from iDCs. iDC adaptations to 3D collagen include increased pro-inflammatory cytokine production and reduced antiviral gene expression in response to HIV-1 infection. Adhesion to a 2D collagen matrix is sufficient to increase iDC deformability, DC-SIGN exposure, and permissivity to HIV-1 infection. Thus, mechano-physical cues of 2D and 3D tissue-like collagen environments regulate iDC function and shape divergent roles during HIV-1 infection.

Amphiphiles Formed from Synthetic DNA-Nanomotifs Mimic the Stepwise Dispersal of Transcriptional Clusters in the Cell Nucleus.

Stem cells exhibit prominent clusters controlling the transcription of genes into RNA. These clusters form by a phase-separation mechanism, and their size and shape are controlled via an amphiphilic effect of transcribed genes. Here, we construct amphiphile-nanomotifs purely from DNA, and we achieve similar size and shape control for phase-separated droplets formed from fully synthetic, self-interacting DNA-nanomotifs. Increasing amphiphile concentrations induce rounding of droplets, prevent droplet fusion, and, at high concentrations, cause full dispersal of droplets. Super-resolution microscopy data obtained from zebrafish embryo stem cells reveal a comparable transition for transcriptional clusters with increasing transcription levels. Brownian dynamics and lattice simulations further confirm that the addition of amphiphilic particles is sufficient to explain the observed changes in shape and size. Our work reproduces key aspects of transcriptional cluster formation in biological cells using relatively simple DNA sequence-programmable nanostructures, opening novel ways to control the mesoscopic organization of synthetic nanomaterials.

Modulating Lipid Membrane Morphology by Dynamic DNA Origami Networks.

Membrane morphology and its dynamic adaptation regulate many cellular functions, which are often mediated by membrane proteins. Advances in DNA nanotechnology have enabled the realization of various protein-inspired structures and functions with precise control at the nanometer level, suggesting a viable tool to artificially engineer membrane morphology. In this work, we demonstrate a DNA origami cross (DOC) structure that can be anchored onto giant unilamellar vesicles (GUVs) and subsequently polymerized into micrometer-scale reconfigurable one-dimensional (1D) chains or two-dimensional (2D) lattices. Such DNA origami-based networks can be switched between left-handed (LH) and right-handed (RH) conformations by DNA fuels and exhibit potent efficacy in remodeling the membrane curvatures of GUVs. This work sheds light on designing hierarchically assembled dynamic DNA systems for the programmable modulation of synthetic cells for useful applications.

A DNA Segregation Module for Synthetic Cells.

The bottom-up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made toward reliable compartment division, yet mechanisms to segregate the DNA-encoded informational content are still in their infancy. Herein, droplets of DNA Y-motifs are formed by liquid-liquid phase separation. DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y-motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio-temporally controlled DNA segregation in bulk as well as in cell-sized water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice-based theoretical model which mimics the interactions between the DNA Y-motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, can represent a strategy toward a DNA segregation module within bottom-up assembled synthetic cells.

RNA origami: design, simulation and application.

Design strategies for DNA and RNA nanostructures have developed along parallel lines for the past 30 years, from small structural motifs derived from biology to large 'origami' structures with thousands to tens of thousands of bases. With the recent publication of numerous RNA origami structures and improved design methods-even permitting co-transcriptional folding of kilobase-sized structures - the RNA nanotechnolgy field is at an inflection point. Here, we review the key achievements which inspired and enabled RNA origami design and draw comparisons with the development and applications of DNA origami structures. We further present the available computational tools for the design and the simulation, which will be key to the growth of the RNA origami community. Finally, we portray the transition from RNA origami structure to function. Several functional RNA origami structures exist already, their expression in cells has been demonstrated and first applications in cell biology have already been realized. Overall, we foresee that the fast-paced RNA origami field will provide new molecular hardware for biophysics, synthetic biology and biomedicine, complementing the DNA origami toolbox.

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.

Tuning Epithelial Cell-Cell Adhesion and Collective Dynamics with Functional DNA-E-Cadherin Hybrid Linkers.

The binding strength between epithelial cells is crucial for tissue integrity, signal transduction and collective cell dynamics. However, there is no experimental approach to precisely modulate cell-cell adhesion strength at the cellular and molecular level. Here, we establish DNA nanotechnology as a tool to control cell-cell adhesion of epithelial cells. We designed a DNA-E-cadherin hybrid system consisting of complementary DNA strands covalently bound to a truncated E-cadherin with a modified extracellular domain. DNA sequence design allows to tune the DNA-E-cadherin hybrid molecular binding strength, while retaining its cytosolic interactions and downstream signaling capabilities. The DNA-E-cadherin hybrid facilitates strong and reversible cell-cell adhesion in E-cadherin deficient cells by forming mechanotransducive adherens junctions. We assess the direct influence of cell-cell adhesion strength on intracellular signaling and collective cell dynamics. This highlights the scope of DNA nanotechnology as a precision technology to study and engineer cell collectives.

Actomyosin-Assisted Pulling of Lipid Nanotubes from Lipid Vesicles and Cells.

Molecular motors are pivotal for intracellular transport as well as cell motility and have great potential to be put to use outside cells. Here, we exploit engineered motor proteins in combination with self-assembly of actin filaments to actively pull lipid nanotubes from giant unilamellar vesicles (GUVs). In particular, actin filaments are bound to the outer GUV membrane and the GUVs are seeded on a heavy meromyosin-coated substrate. Upon addition of ATP, hollow lipid nanotubes with a length of tens of micrometer are pulled from single GUVs due to the motor activity. We employ the same mechanism to pull lipid nanotubes from different types of cells. We find that the length and number of nanotubes critically depends on the cell type, whereby suspension cells form bigger networks than adherent cells. This suggests that molecular machines can be used to exert forces on living cells to probe membrane-to-cortex attachment.

Genotype-phenotype mapping with polyominos made from DNA origami tiles.

The correlation between genetic information and characteristics of a living cell-its genotype and its phenotype-constitutes the basis of genetics. Here, we experimentally realize a primitive form of genotype-phenotype mapping with DNA origami. The DNA origami can polymerize into two-dimensional lattices (phenotype) via blunt-end stacking facilitated by edge staples at the seam of the planar DNA origami. There are 80 binding positions for edge staples, which allow us to translate an 80-bit long binary code (genotype) onto the DNA origami. The presence of an edge staple thus corresponds to a "1" and its absence to a "0." The interactions of our DNA-based system can be reproduced by a polyomino model. Polyomino growth simulations qualitatively reproduce our experimental results. We show that not only the absolute number of base stacks but also their sequence position determine the cluster size and correlation length of the orientation of single DNA origami within the cluster. Importantly, the mutation of a few bits can result in major morphology changes of the DNA origami cluster, while more often, major sequence changes have no impact. Our experimental realization of a correlation between binary information ("genotype") and cluster morphology ("phenotype") thus reproduces key properties of genotype-phenotype maps known from living systems.

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.

Evolution and Single-Droplet Analysis of Fuel-Driven Compartments by Droplet-Based Microfluidics.

Active droplets are a great model for membraneless organelles. However, the analysis of these systems remains challenging and is often limited due to the short timescales of their kinetics. We used droplet-based microfluidics to encapsulate a fuel-driven cycle that drives phase separation into coacervate-based droplets to overcome this challenge. This approach enables the analysis of every coacervate-based droplet in the reaction container throughout its lifetime. We discovered that the fuel concentration dictates the formation of the coacervate-based droplets and their properties. We observed that coacervate-based droplets grow through fusion, decay simultaneously independent of their volume, and shrinkage rate scales with their initial volume. This method helps to further understand the regulation of membraneless organelles, and we believe the analysis of individual coacervate-based droplets enables future selection- or evolution-based studies.

Controlling aggregation of cholesterol-modified DNA nanostructures.

DNA nanotechnology allows for the design of programmable DNA-built nanodevices which controllably interact with biological membranes and even mimic the function of natural membrane proteins. Hydrophobic modifications, covalently linked to the DNA, are essential for targeted interfacing of DNA nanostructures with lipid membranes. However, these hydrophobic tags typically induce undesired aggregation eliminating structural control, the primary advantage of DNA nanotechnology. Here, we study the aggregation of cholesterol-modified DNA nanostructures using a combined approach of non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering, confocal microscopy and atomistic molecular dynamics simulations. We show that the aggregation of cholesterol-tagged ssDNA is sequence-dependent, while for assembled DNA constructs, the number and position of the cholesterol tags are the dominating factors. Molecular dynamics simulations of cholesterol-modified ssDNA reveal that the nucleotides wrap around the hydrophobic moiety, shielding it from the environment. Utilizing this behavior, we demonstrate experimentally that the aggregation of cholesterol-modified DNA nanostructures can be controlled by the length of ssDNA overhangs positioned adjacent to the cholesterol. Our easy-to-implement method for tuning cholesterol-mediated aggregation allows for increased control and a closer structure-function relationship of membrane-interfacing DNA constructs - a fundamental prerequisite for employing DNA nanodevices in research and biomedicine.

Dynamic Actuation of DNA-Assembled Plasmonic Nanostructures in Microfluidic Cell-Sized Compartments.

Molecular motor proteins form the basis of cellular dynamics. Recently, notable efforts have led to the creation of their DNA-based mimics, which can carry out complex nanoscale motion. However, such functional analogues have not yet been integrated or operated inside synthetic cells toward the goal of realizing artificial biological systems entirely from the bottom-up. In this Letter, we encapsulate and actuate DNA-assembled dynamic nanostructures inside cell-sized microfluidic compartments. These encapsulated DNA nanostructures not only exhibit structural reconfigurability owing to their pH-sensitive molecular switches upon external stimuli but also possess optical feedback enabled by the integrated plasmonic probes. In particular, we demonstrate the power of microfluidic compartmentalization for achieving on-chip plasmonic enantiomer separation and substrate filtration. Our work exemplifies that the two unique tools, droplet-based microfluidics and DNA technology, offering high precision on the microscale and nanoscale, respectively, can be brought together to greatly enrich the complexity and diversity of functional synthetic systems.

Division and Regrowth of Phase-Separated Giant Unilamellar Vesicles*.

Success in the bottom-up assembly of synthetic cells will depend on strategies for the division of protocellular compartments. Here, we describe the controlled division of phase-separated giant unilamellar lipid vesicles (GUVs). We derive an analytical model based on the vesicle geometry, which makes four quantitative predictions that we verify experimentally. We find that the osmolarity ratio required for division is 2 , independent of the GUV size, while asymmetric division happens at lower osmolarity ratios. Remarkably, we show that a suitable osmolarity change can be triggered by water evaporation, enzymatic decomposition of sucrose or light-triggered uncaging of CMNB-fluorescein. The latter provides full spatiotemporal control, such that a target GUV undergoes division whereas the surrounding GUVs remain unaffected. Finally, we grow phase-separated vesicles from single-phased vesicles by targeted fusion of the opposite lipid type with programmable DNA tags to enable subsequent division cycles.

DNA Nanotechnology for Building Sensors, Nanopores and Ion-Channels.

DNA nanotechnology has revolutionised the capabilities to shape and control three-dimensional structures at the nanometre scale. Designer sensors, nanopores and ion-channels built from DNA have great potential for both cross-disciplinary research and applications. Here, we introduce the concept of structural DNA nanotechnology, including DNA origami, and give an overview of the work flow from design to assembly, characterisation and application of DNA-based functional systems. Chemical functionalisation of DNA has opened up pathways to transform static DNA structures into dynamic nanomechanical sensors. We further introduce nanopore sensing as a powerful label-free single-molecule technique and discuss how it can benefit from DNA nanotechnology. Especially exciting is the possibility to create membrane-inserted DNA nanochannels that mimic their protein-based natural counterparts in form and function. In this chapter we review the status quo of DNA sensors, nanopores and ion channels, highlighting opportunities and challenges for their future development.

Ion Channels Made from a Single Membrane-Spanning DNA Duplex.

Because of their hollow interior, transmembrane channels are capable of opening up pathways for ions across lipid membranes of living cells. Here, we demonstrate ion conduction induced by a single DNA duplex that lacks a hollow central channel. Decorated with six porpyrin-tags, our duplex is designed to span lipid membranes. Combining electrophysiology measurements with all-atom molecular dynamics simulations, we elucidate the microscopic conductance pathway. Ions flow at the DNA-lipid interface as the lipid head groups tilt toward the amphiphilic duplex forming a toroidal pore filled with water and ions. Ionic current traces produced by the DNA-lipid channel show well-defined insertion steps, closures, and gating similar to those observed for traditional protein channels or synthetic pores. Ionic conductances obtained through simulations and experiments are in excellent quantitative agreement. The conductance mechanism realized here with the smallest possible DNA-based ion channel offers a route to design a new class of synthetic ion channels with maximum simplicity.

DNA-Tile Structures Induce Ionic Currents through Lipid Membranes.

Self-assembled DNA nanostructures have been used to create man-made transmembrane channels in lipid bilayers. Here, we present a DNA-tile structure with a nominal subnanometer channel and cholesterol-tags for membrane anchoring. With an outer diameter of 5 nm and a molecular weight of 45 kDa, the dimensions of our synthetic nanostructure are comparable to biological ion channels. Because of its simple design, the structure self-assembles within a minute, making its creation scalable for applications in biology. Ionic current recordings demonstrate that the tile structures enable ion conduction through lipid bilayers and show gating and voltage-switching behavior. By demonstrating the design of DNA-based membrane channels with openings much smaller than that of the archetypical six-helix bundle, our work showcases their versatility inspired by the rich diversity of natural membrane components.

Membrane localization of actin filaments stabilizes giant unilamellar vesicles against external deforming forces.

Actin organization is crucial for establishing cell polarity, which influences processes such as directed cell motility and division. Despite its critical role in living organisms, achieving similar polarity in synthetic cells remains challenging. In this study, we employ a bottom-up approach to investigate how molecular crowders facilitate the formation of cortex-like actin networks and how these networks localize and organize based on membrane shape. Using giant unilamellar vesicles (GUVs) as models for cell membranes, we show that actin filaments can arrange along the membrane to form cortex-like structures. Notably, this organization is achieved using only actin and crowders as a minimal set of components. We utilize surface micropatterning to examine actin filament organization in deformed GUVs adhered to various pattern shapes. Our findings indicate that at the periphery of spherical GUVs, actin bundles align along the membrane. However, in highly curved regions of adhered GUVs, actin bundles avoid crossing the highly curved edges perpendicular to the adhesion site and instead remain in the lower curved regions by aligning parallel to the micropatterned surface. Furthermore, the actin bundles increase the stiffness of the GUVs, effectively counteracting strong deformations when GUVs adhere to micropatterns. This finding is corroborated by real-time deformability cytometry on GUVs with synthetic actin cortices. By precisely manipulating the shape of GUVs, our study provides a minimal system to investigate the interplay between actin structures and the membrane. Our findings provide insights into the spatial organization of actin structures within crowded environments, specifically inside GUVs that resemble the size and shape of cells. This study advances our understanding of actin network organization and functionality within cell-sized compartments.

Minimal-Invasive 3D Laser Printing of Microimplants in Organismo.

Multi-photon 3D laser printing has gathered much attention in recent years as a means of manufacturing biocompatible scaffolds that can modify and guide cellular behavior in vitro. However, in vivo tissue engineering efforts have been limited so far to the implantation of beforehand 3D printed biocompatible scaffolds and in vivo bioprinting of tissue constructs from bioinks containing cells, biomolecules, and printable hydrogel formulations. Thus, a comprehensive 3D laser printing platform for in vivo and in situ manufacturing of microimplants raised from synthetic polymer-based inks is currently missing. Here, a platform for minimal-invasive manufacturing of microimplants directly in the organism is presented by one-photon photopolymerization and multi-photon 3D laser printing. Employing a commercially available elastomeric ink giving rise to biocompatible synthetic polymer-based microimplants, first applicational examples of biological responses to in situ printed microimplants are demonstrated in the teleost fish Oryzias latipes and in embryos of the fruit fly Drosophila melanogaster. This provides a framework for future studies addressing the suitability of inks for in vivo 3D manufacturing. The platform bears great potential for the direct engineering of the intricate microarchitectures in a variety of tissues in model organisms and beyond.

Triggered contraction of self-assembled micron-scale DNA nanotube rings.

Contractile rings are formed from cytoskeletal filaments during cell division. Ring formation is induced by specific crosslinkers, while contraction is typically associated with motor protein activity. Here, we engineer DNA nanotubes and peptide-functionalized starPEG constructs as synthetic crosslinkers to mimic this process. The crosslinker induces bundling of ten to hundred DNA nanotubes into closed micron-scale rings in a one-pot self-assembly process yielding several thousand rings per microliter. Molecular dynamics simulations reproduce the detailed architectural properties of the DNA rings observed in electron microscopy. Theory and simulations predict DNA ring contraction - without motor proteins - providing mechanistic insights into the parameter space relevant for efficient nanotube sliding. In agreement between simulation and experiment, we obtain ring contraction to less than half of the initial ring diameter. DNA-based contractile rings hold promise for an artificial division machinery or contractile muscle-like materials.