Designer peptide–DNA cytoskeletons regulate the function of synthetic cells

nature chemistry Article https://doi.org/10.1038/s41557-024-01509-w Designer peptide–DNA cytoskeletons regulate the function of synthetic cells Received: 13 June 2022 Accepted: 15 March 2024 Published online: xx xx xxxx Check for updates Margaret L. Daly 1,2, Kengo Nishi1,2, Stephen J. Klawa Yuan Gao1 & Ronit Freeman 1 , Kameryn Y. Hinton 1 The bottom-up engineering of artificial cells requires a reconfigurable cytoskeleton that can organize at distinct locations and dynamically modulate its structural and mechanical properties. Here, inspired by the vast array of actin-binding proteins and their ability to reversibly crosslink or bundle filaments, we have designed a library of peptide–DNA crosslinkers varying in length, valency and geometry. Peptide filaments conjoint through DNA hybridization give rise to tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and complementary DNA handles allow for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide–DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. Altogether, the modular design of peptide–DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells. Engineering synthetic cytoskeletons is essential for the bottom-up construction of artificial cells. The cellular cytoskeleton consists of hierarchical and dynamic polymers that function as scaffolding components of cells and drive vital processes, including cell division, motility and morphogenesis1. These functionalities are governed by the spatial organization of the cytoskeletal components inside cells and in contact with membranes. The cytoskeleton’s ability to transition between various architectures, ranging from filament networks to aligned bundles or spindles, is regulated by numerous associating proteins2,3. These proteins regulate the nucleation, elongation, branching, severing, capping, bundling and crosslinking of filaments to shape cells1,4,5. In vitro systems using purified proteins have expanded our understanding of how cytoskeletal filaments and their associating proteins shape cells. Purified proteins reconstituted within or on cell-sized vesicles and droplets have been used to explore the effect of actin organization on cell shape6. For example, the crosslinking of actin with fascin, actinin or filamin in giant unilamellar vesicles was investigated to understand how different bundled actin assemblies induce membrane deformation7–10. Recently, engineering of the synthetic cytoskeleton has been proposed as a pathway to generate artificial cells11. While various building blocks form filaments and networks in the bulk, including polymers12, small molecules13, carbon nanotubes14, peptides13,15 and DNA16–18, assembly in cell-like confinement has so far mainly relied on DNA material19–23. The bundling of DNA nanofilaments in protocells has been achieved by adding crowding agents20 or salt23, which is challenging to reverse or fine-tune. Another synthetic challenge is to recruit structures to the periphery or lumen of droplets. While cholesterol20,23 or lipid tail24 modifications have been used to localize structures to membranes, a more controlled spatial localization would enable cytoskeletal–membrane mechanical and biochemical crosstalk towards constructing deformable and responsive artificial cells. Peptides are a less used, yet promising building block for the construction of synthetic cytoskeletons. The rational design of peptides Department of Applied Physical Sciences, University of North Carolina, Chapel Hill, NC, USA. 2These authors contributed equally: Margaret L. Daly, Kengo Nishi. e-mail: 1 Nature Chemistry , 1 Article https://doi.org/10.1038/s41557-024-01509-w Actin cytoskeleton Actin-inspired cytoskeleton Crosslinking proteins Peptide–DNA crosslinkers Self-assembled cytoskeleton Fig. 1 | Peptide–DNA nanotechnology for the construction of synthetic cytoskeletons. Peptide–DNA filaments crosslinked via programmable complementary DNA interactions (middle), mimicking actin (left), and its associated proteins. The tunable organization of the peptide–DNA cytoskeleton in cell-sized confinement (right) guides the functions of artificial cells. assembling into diverse structures across length scales has been extensively studied in the bulk25,26. Yet, only a handful of peptide-based systems have been realized in cell-like confinement24,27–31. Peptide filament assembly in water-in-oil droplets has been triggered by pH or salt24,28,29, and short self-assembled peptides have been shown to stabilize water-in-oil droplets30,32,33. Unleashing the full potential of peptide-based self-assembled systems in confinement will introduce unique properties and capabilities into synthetic cells. In this study, we married peptide self-assembly with DNA programmability to realize a synthetic cytoskeleton in droplets (Fig. 1). Inspired by actin-binding proteins, we rationally designed peptide–DNA crosslinkers with varying sequence, length, valency and geometry. We show here how filamentous peptides conjoined through DNA hybridization form tactoid-shaped bundles and networks with tunable aspect ratios and mechanics. When confined within cell-sized water-in-oil droplets, distinct structures are driven to spatially localize in the cortex or lumen, depending on the crosslinker attributes, and the extent of bundling tunes the mobility of intradroplet payloads from water-like to arrested. Finally, we show how different crosslinkers orchestrate changes in the cellular shape of lipid-encased droplets. Our programmable peptide–DNA nanotechnology approach is a powerful platform towards the construction of functional, fully artificial cells. Results and discussion Peptide–DNA constructs crosslinking cytoskeletal filaments Cytoskeletal actin filaments associate with actin-binding crosslinkers of different geometries and flexibilities to form mesoscale functional structures, such as stress fibres, filopodia and the cell cortex1. For instance, fascin organizes actin filaments into bundles to regulate cell migration, while filamin crosslinks actin to form networks in the cell cortex3,34. Given the essential role of actin-binding proteins in regulating various cytoskeletal arrangements, we hypothesized that mimetic crosslinkers will extend the functionality of synthetic filamentous systems to yield reconfigurable cytoskeletal superstructures. To test this, we used the emerging class of peptide–DNA materials, Nature Chemistry uniquely integrating peptide self-assembly with DNA programmability35,36. Previously, Stupp and co-workers sh (...truncated)