Defined DNA-Mediated Assemblies of Gene-Expressing Giant Unilamellar Vesicles

Abstract

The technological aspects of artificial vesicles as prominent cell mimics are evolving toward higher-order assemblies of functional vesicles with tissuelike architectures. Here, we demonstrate the spatially controlled DNA-directed bottom-up synthesis of complex microassemblies and macroassemblies of giant unilamellar vesicles functionalized with a basic cellular machinery to express green fluorescent protein and specified neighbor-to-neighbor interactions. We show both that the local and programmable DNA pairing rules on the nanoscale are able to direct the microscale vesicles into macroscale soft matter assemblies and that the highly sensitive gene-expression machinery remains intact and active during multiple experimental steps. An in silico model recapitulates the experiments performed in vitro and covers additional experimental setups highlighting the parameters that control the DNA-directed bottom-up synthesis of higher-order self-assembled structures. The controlled assembly of a functional vesicle matrix may be useful not only as simplified natural tissue mimics but also as artificial scaffolds that could interact and support living cells.

From Cells as Computation to Cells as Apps

“…For example, it has been reported that lipid compartments can form clusters following simple adhesive forces [28,14]. Protein synthesis is still possible in such assemblies [27], and the idea of prototissue has been put forward, as supported by some pioneer work on membrane-to-membrane connective mojeties [49,42].…”

Formalizing Modularization and Data Hiding in Synthetic Biology

“…1 the spatially controlled DNA-directed bottom-up synthesis of complex tissue-like assemblies composed of artificial vesicles was implemented. In addition to the specified neighbor-to-neighbor interactions DNA also provided the genetic blueprint for the synthesis of functional proteins [30] (Fig. 2).…”

“…Scale bars: 1000 µm (b, e, h, l), 50 µm (c, f, j, m). Fluorescence micrographs b, c, e, f modified from Hadorn et al [30]; h, j, l, m modified from Hadorn et al [31].…”

“…Other studies employed supramolecular nanoscale and microscale compartments as "nano-bioreactors" that result from the non-covalent interaction of molecules (i.e., primary self-assembly process) [1,[53][54][55]63]. Several protocols have been established to control the self-assembly of these compartments into higher-order structures (i.e., secondary self-assembly process) [3,30,32] as well as to induce budding [10] and fusion [17,61,75]. Even protocols for internal compartmentalization of artificial vesicles have been reported [9,11,36,40,43,86].…”

“…Assemblies of vesicles have been proposed for engineered nano-bioreactors [35], as multicomponent or multifunctional drug delivery systems [82], and as starting point for the synthesis of primitive cell communities [15]. In order to converge artificial vesicles and natural cells, single vesicles either solitary [34,55,56] or incorporated in higher-order structures [30] were functionalized with artificial genetic programs and cellular components to transfer genetic blueprints into functional proteins. Oil-in-water emulsion compartments, on the other hand, offer robust, configurable, and recyclable soft colloid systems with interesting properties, and were proposed for the fabrication of delivery systems for lipophilic substances and advanced synthetic materials with adaptable properties [7].…”