Bottom-Up Assembly of Synthetic Cells with a DNA Cytoskeleton

Abstract

Cytoskeletal elements, like actin and myosin, have been reconstituted inside lipid vesicles toward the vision to reconstruct cells from the bottom up. Here, we realize the de novo assembly of entirely artificial DNA-based cytoskeletons with programmed multifunctionality inside synthetic cells. Giant unilamellar lipid vesicles (GUVs) serve as cell-like compartments, in which the DNA cytoskeletons are repeatedly and reversibly assembled and disassembled with light using the cis – trans isomerization of an azobenzene moiety positioned in the DNA tiles. Importantly, we induced ordered bundling of hundreds of DNA filaments into more rigid structures with molecular crowders. We quantify and tune the persistence length of the bundled filaments to achieve the formation of ring-like cortical structures inside GUVs, resembling actin rings that form during cell division. Additionally, we show that DNA filaments can be programmably linked to the compartment periphery using cholesterol-tagged DNA as a linker. The linker concentration determines the degree of the cortex-like network formation, and we demonstrate that the DNA cortex-like network can deform GUVs from within. All in all, this showcases the potential of DNA nanotechnology to mimic the diverse functions of a cytoskeleton in synthetic cells.

Network topology–directed design of molecular CPU for cell-like dynamic information processing

“…Future studies may include integrating more processing modules for biomedical applications in response to dynamic stimuli and improving the stability of the ACs for prolonged blood circulation time. A possible strategy to stabilize the structure of AC is to build artificial cytoskeleton inside or outside the AC membrane to reduce rupture during long-term circulation ( 60 ). For the equipped DNA NP and encapsulated DNA network, nuclease-resistant nonnatural nucleic acids such as l -DNA can be used to enhance system robustness.…”

Mechanochemical signal transduction in synthetic cells

“…However, apart from molecular substrate exchange, a mechanochemical connection to intracellular downstream functions within synthetic cellular compartments is missing. Concomitantly, DNA nanotubes as structural and functional mimics of cytoskeletons have been reconstituted within cell-sized water-in-oil droplets 17,18 and recently also in GUVs 5 . Here, we link filament disassembly and cytoskeletal remodeling within GUVs to transmembrane properties of DNA origami and chemical signalling from the outside of GUVs reminiscent of focal adhesions within natural cells 19 .…”

“…To employ the engineered DOSUs for chemical signalling (Feature (iii)), we interfaced our DNA pores with DNA-based mimics of cytoskeletons. We used DNA double-crossover tiles 27 which self-assemble into hollow DNA filaments and encapsulated them into GUVs 5 . The DNA filaments were modified with single-stranded overhangs so that they can be disassembled by toehold-mediated strand displacement 18 .…”

“…The incorporation of membrane-spanning structures to guide cellular function and to enable transmembrane signalling is therefore a critical aim for bottom-up synthetic biology 2,3,4 . Here, we design membrane-spanning DNA origami signalling units (DOSUs) and mechanically couple them to DNA cytoskeletons 5 encapsulated within giant unilamellar vesicles (GUVs). We verify the assembly and incorporation of the DOSUs into the GUV membranes and achieve their clustering upon external stimulation.…”

“…(DOSUs) and mechanically couple them to DNA cytoskeletons 5 encapsulated within giant unilamellar vesicles (GUVs). We verify the assembly and incorporation of the DOSUs into the GUV membranes and achieve their clustering upon external stimulation.…”