Patterning DNA Origami on Membranes Through Protein Self-Organization

Gür F.N., Kempter S., Schueder F., Sikeler C., Urban M.J., Jungmann R., Nickels P.C., Liedl T.

The design of dynamic, reconfigurable devices is crucial for the bottom-up construction of artificial biological systems. DNA can be used as an engineering material for the de-novo design of such dynamic devices. A self-assembled DNA origami switch is presented that uses the transition from double- to single-stranded DNA and vice versa to create and annihilate an entropic force that drives a reversible conformational change inside the switch. It is distinctively demonstrated that a DNA single-strand that is extended with 0.34 nm per nucleotide – the extension this very strand has in the double-stranded configuration – exerts a contractive force on its ends leading to large-scale motion. The operation of this type of switch is demonstrated via transmission electron microscopy, DNA-PAINT super-resolution microscopy and darkfield microscopy. The work illustrates the intricate and sometimes counter-intuitive forces that act in nanoscale physical systems that operate in fluids.

Reuther C., Catalano R., Salhotra A., Vemula V., Korten T., Diez S., Månsson A.

Abstract Over the last 25 years, extensive progress has been made in developing a range of nanotechnological applications where cytoskeletal filaments and molecular motors are key elements. This includes novel, highly miniaturized lab on a chip systems for biosensing, nanoseparation etc but also new materials and parallel computation devices for solving otherwise intractable mathematical problems. For such approaches, both actin-based and microtubule-based cytoskeletal systems have been used. However, in accordance with their different cellular functions, actin filaments and microtubules have different properties and interaction kinetics with molecular motors. Therefore, the two systems obviously exhibit different advantages and encounter different challenges when exploited for applications. Specifically, the achievable filament velocities, the capability to guide filaments along nanopatterned tracks and the capability to attach and transport cargo differ between actin- and microtubule-based systems. Our aim here is to systematically elucidate these differences to facilitate design of new devices and optimize future developments. We first review the cellular functions and the fundamental physical and biochemical properties of actin filaments and microtubules. In this context we also consider their interaction with molecular motors and other regulatory proteins that are of relevance for applications. We then relate these properties to the advantages and challenges associated with the use of each of the motor-filament systems for different tasks. Finally, fundamental properties are considered in relation to some of the most interesting future development paths e.g. in biosensing and biocomputation.

Fragasso A., De Franceschi N., Stömmer P., van der Sluis E.O., Dietz H., Dekker C.

Molecular traffic across lipid membranes is a vital process in cell biology that involves specialized biological pores with a great variety of pore diameters, from fractions of a nanometer to >30 nm. Creating artificial membrane pores covering similar size and complexity will aid the understanding of transmembrane molecular transport in cells, while artificial pores are also a necessary ingredient for synthetic cells. Here, we report the construction of DNA origami nanopores that have an inner diameter as large as 30 nm. We developed methods to successfully insert these ultrawide pores into the lipid membrane of giant unilamellar vesicles (GUVs) by administering the pores concomitantly with vesicle formation in an inverted-emulsion cDICE technique. The reconstituted pores permit the transmembrane diffusion of large macromolecules, such as folded proteins, which demonstrates the formation of large membrane-spanning open pores. The pores are size selective, as dextran molecules with a diameter up to 28 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modeling of the GFP influx rate, we find that up to hundreds of pores can be functionally reconstituted into a single GUV. Our technique bears great potential for applications across different fields from biomimetics, to synthetic biology, to drug delivery.

Ramirez-Diaz D.A., Merino-Salomón A., Meyer F., Heymann M., Rivas G., Bramkamp M., Schwille P.

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner. During bacterial cell division, the protein FtsZ is the main component of the contractile ring, though how precisely FtsZ treadmilling and its ability to deform membranes cooperate are unclear. Here, the authors show that dynamic FtsZ may deform lipid membranes via torsional stress that may provide sufficient force to constrict membranes in vivo and in vitro.

Kern N., Dong R., Douglas S.M., Vale R.D., Morrissey M.A.

Macrophages destroy pathogens and diseased cells through Fcγ receptor (FcγR)-driven phagocytosis of antibody-opsonized targets. Phagocytosis requires activation of multiple FcγRs, but the mechanism controlling the threshold for response is unclear. We developed a DNA origami-based engulfment system that allows precise nanoscale control of the number and spacing of ligands. When the number of ligands remains constant, reducing ligand spacing from 17.5 nm to 7 nm potently enhances engulfment, primarily by increasing efficiency of the engulfment-initiation process. Tighter ligand clustering increases receptor phosphorylation, as well as proximal downstream signals. Increasing the number of signaling domains recruited to a single ligand-receptor complex was not sufficient to recapitulate this effect, indicating that clustering of multiple receptors is required. Our results suggest that macrophages use information about local ligand densities to make critical engulfment decisions, which has implications for the mechanism of antibody-mediated phagocytosis and the design of immunotherapies.

Berger R.M., Weck J.M., Kempe S.M., Hill O., Liedl T., Rädler J.O., Monzel C., Heuer‐Jungemann A.

Engelen W., Dietz H.

DNA origami enables the bottom-up construction of chemically addressable, nanoscale objects with user-defined shapes and tailored functionalities. As such, not only can DNA origami objects be used to improve existing experimental methods in biophysics, but they also open up completely new avenues of exploration. In this review, we discuss basic biophysical concepts that are relevant for prospective DNA origami users. We summarize biochemical strategies for interfacing DNA origami with biomolecules of interest. We describe various applications of DNA origami, emphasizing the added value or new biophysical insights that can be generated: rulers and positioning devices, force measurement and force application devices, alignment supports for structural analysis for biomolecules in cryogenic electron microscopy and nuclear magnetic resonance, probes for manipulating and interacting with lipid membranes, and programmable nanopores. We conclude with some thoughts on so-far little explored opportunities for using DNA origami in more complex environments such as the cell or even organisms.

Kretschmer S., Heermann T., Tassinari A., Glock P., Schwille P.

The formation of large-scale patterns through molecular self-organization is a basic principle of life. Accordingly, the engineering of protein patterns and gradients is of prime relevance for synthetic biology. As a paradigm for such pattern formation, the bacterial MinDE protein system is based on self-organization of the ATPase MinD and ATPase-activating protein MinE on lipid membranes. Min patterns can be tightly regulated by tuning physical or biochemical parameters. Among the biochemically engineerable modules, MinD's membrane targeting sequence, despite being a key regulating element, has received little attention. Here we attempt to engineer patterns by modulating the membrane affinity of MinD. Unlike the traveling waves or stationary patterns commonly observed in vitro on flat supported membranes, standing-wave oscillations emerge upon elongating MinD's membrane targeting sequence via rationally guided mutagenesis. These patterns are capable of forming gradients and thereby spatially target co-reconstituted downstream proteins, highlighting their functional potential in designing new life-like systems.

Shaw T.R., Ghosh S., Veatch S.L.

Lateral organization in the plane of the plasma membrane is an important driver of biological processes. The past dozen years have seen increasing experimental support for the notion that lipid organization plays an important role in modulating this heterogeneity. Various biophysical mechanisms rooted in the concept of liquid–liquid phase separation have been proposed to explain diverse experimental observations of heterogeneity in model and cell membranes with distinct but overlapping applicability. In this review, we focus on the evidence for and the consequences of the hypothesis that the plasma membrane is poised near an equilibrium miscibility critical point. Critical phenomena explain certain features of the heterogeneity observed in cells and model systems but also go beyond heterogeneity to predict other interesting phenomena, including responses to perturbations in membrane composition.

Ramm B., Goychuk A., Khmelinskaia A., Blumhardt P., Eto H., Ganzinger K.A., Frey E., Schwille P.

The healthy growth and maintenance of a biological system depends on the precise spatial organization of molecules within the cell through the dissipation of energy. Reaction–diffusion mechanisms can facilitate this organization, as can directional cargo transport orchestrated by motor proteins, by relying on specific protein interactions. However, transport of material through the cell can also be achieved by active processes based on non-specific, purely physical mechanisms, a phenomenon that remains poorly explored. Here, using a combined experimental and theoretical approach, we discover and describe a hidden function of the Escherichia coli MinDE protein system: in addition to forming dynamic patterns, this system accomplishes the directional active transport of functionally unrelated cargo on membranes. Remarkably, this mechanism enables the sorting of diffusive objects according to their effective size, as evidenced using modular DNA origami–streptavidin nanostructures. We show that the diffusive fluxes of MinDE and non-specific cargo couple via density-dependent friction. This non-specific process constitutes a diffusiophoretic mechanism, as yet unknown in a cell biology setting. This nonlinear coupling between diffusive fluxes could represent a generic physical mechanism for establishing intracellular organization. Protein oscillations linked to cell division in Escherichia coli are shown to localize unrelated molecules on the cell membrane via a diffusiophoretic mechanism, in which an effective friction fosters cargo transport along the fluxes set up by the proteins.

Eto H., Franquelim H.G., Heymann M., Schwille P.

This paper outlines a robust method to template biological membranes in 3D geometries using micron-scale 3D printing. Dynamic protein systems were reconstituted in vitro and their self-organization was observed in response to the 3D geometry.

Hellmeier J., Platzer R., Eklund A.S., Schlichthaerle T., Karner A., Motsch V., Schneider M.C., Kurz E., Bamieh V., Brameshuber M., Preiner J., Jungmann R., Stockinger H., Schütz G.J., Huppa J.B., et. al.

Significance The nanoscale organization of ligands and receptors is critical for cellular communication yet inherently challenging to investigate. We have here devised a DNA origami-based biointerface which allows the experimenter to adjust protein distances with nanometer precision as a means to enhance or disturb signaling while being responsive to large-scale reorganization processes during cell activation. Applying this biointerface to study the spatial requirements of T cell activation, we find that the smallest signaling-competent receptor unit consists of two stably ligated T cell receptors (TCRs) within a distance of 20 nanometers. Spatial organization of the physiological ligand pMHC, however, is not a relevant parameter of antigen-mediated T cell activation, as single, well-isolated pMHC molecules efficiently stimulate T cells.

van de Schoot R., Depaoli S., King R., Kramer B., Märtens K., Tadesse M.G., Vannucci M., Gelman A., Veen D., Willemsen J., Yau C.

Bayesian statistics is an approach to data analysis based on Bayes’ theorem, where available knowledge about parameters in a statistical model is updated with the information in observed data. The background knowledge is expressed as a prior distribution and combined with observational data in the form of a likelihood function to determine the posterior distribution. The posterior can also be used for making predictions about future events. This Primer describes the stages involved in Bayesian analysis, from specifying the prior and data models to deriving inference, model checking and refinement. We discuss the importance of prior and posterior predictive checking, selecting a proper technique for sampling from a posterior distribution, variational inference and variable selection. Examples of successful applications of Bayesian analysis across various research fields are provided, including in social sciences, ecology, genetics, medicine and more. We propose strategies for reproducibility and reporting standards, outlining an updated WAMBS (when to Worry and how to Avoid the Misuse of Bayesian Statistics) checklist. Finally, we outline the impact of Bayesian analysis on artificial intelligence, a major goal in the next decade. This Primer on Bayesian statistics summarizes the most important aspects of determining prior distributions, likelihood functions and posterior distributions, in addition to discussing different applications of the method across disciplines.

Franquelim H.G., Dietz H., Schwille P.

Reversible MgCl2-induced blunt-end polymerization of membrane-bound straight DNA origami monomers into filaments leads to protruding deformations on freestanding lipid membranes.

Wang W., Arias D.S., Deserno M., Ren X., Taylor R.E.

DNA nanotechnology has proven exceptionally apt at probing and manipulating biological environments as it can create nanostructures of almost arbitrary shape that permit countless types of modifications, all while being inherently biocompatible. Emergent areas of particular interest are applications involving cellular membranes, but to fully explore the range of possibilities requires interdisciplinary knowledge of DNA nanotechnology, cell and membrane biology, and biophysics. In this review, we aim for a concise introduction to the intersection of these three fields. After briefly revisiting DNA nanotechnology, as well as the biological and mechanical properties of lipid bilayers and cellular membranes, we summarize strategies to mediate interactions between membranes and DNA nanostructures, with a focus on programmed delivery onto, into, and through lipid membranes. We also highlight emerging applications, including membrane sculpting, multicell self-assembly, spatial arrangement and organization of ligands and proteins, biomechanical sensing, synthetic DNA nanopores, biological imaging, and biomelecular sensing. Many critical but exciting challenges lie ahead, and we outline what strikes us as promising directions when translating DNA nanostructures for future in vitro and in vivo membrane applications.

Patil S., Suleman S., Anzar N., Narang J., Pilloton R., Timur S., Guler Celik E., Pundir C.S., Shukla S.K.

Biosensors are widely used across industries such as healthcare, food safety, and environmental monitoring, offering high stability and sensitivity compared to conventional methods. Recently, origami—the art of folding 2D structures into 3D forms—has emerged as a valuable approach in biosensor development, enabling the creation of shape-changing devices. These origami-based biosensors are particularly useful in precision medicine, rapid diagnostics, and resource-limited settings, offering affordable, highly precise, and portable solutions with diverse applications. Paper and biological substrates like DNA have been integrated with origami techniques to develop biosensors with enhanced functionality. The incorporation of aptamer origami into both paper and DNA biosensors further increases sensitivity and specificity for target detection. The concept of paper-based origami biosensors originated from using paper as a platform for biological assays, leading to significant advancements in design and functionality. These devices employ folding techniques to create channels and wells for manipulating samples and detecting target molecules through reactions with specific reagents. Similarly, DNA origami, introduced in 2006, has revolutionized biosensors by enabling the creation of precise molecular systems with tunable properties. Paper-based and DNA origami biosensors have immense potential to transform biosensing technologies in healthcare, food safety, and environmental monitoring. This review explores diverse origami-based biosensor techniques and their applications, including the role of aptamer origami in paper and DNA biosensors.

Gavrilović S., Brüggenthies G.A., Weck J.M., Heuer‐Jungemann A., Schwille P.

AbstractNanofabrication has experienced a big boost with the invention of DNA origami, enabling the production and assembly of complex nanoscale structures that may be able to unlock fully new functionalities in biology and beyond. The remarkable precision with which these structures can be designed and produced is, however, not yet matched by their assembly dynamics, which can be extremely slow, particularly when attached to biological templates, such as membranes. Here, the rapid and controlled formation of DNA origami lattices on the scale of hundreds of micrometers in as little as 30 minutes is demonstrated, utilizing active patterning by the E.coli Min protein system, thereby yielding a remarkable improvement over conventional passive diffusion‐based assembly methods. Various patterns, including spots, inverse spots, mazes, and meshes can be produced at different scales, tailored through the shape and density of the assembled structures. The differential positioning accomplished by Min‐induced diffusiophoresis even allows the introduction of “pseudo‐colors”, i.e., complex core–shell patterns, by simultaneously patterning different DNA origami species. Beyond the targeted functionalization of biological surfaces, this approach may also be promising for applications in plasmonics, catalysis, and molecular sensing.