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Engineering Encapsulated Synthetic Cytoskeletal Dynamics via Nucleic Acid Nanotechnology and Genetic Circuits

Creative Commons 'BY-NC' version 4.0 license

Programmable, synthetic cells have applications in sensing and drug-delivery. Currently, development of synthetic cell components focuses on compartmentalization, and on developing the minimal machinery to carry out different cellular processes. In native cells, cytoskeletal filaments are a key structure for cell division, motility, and intra-cellular transport. Harnessing these filaments for use in synthetic systems is limited by the complexity of the dynamic behavior of the filaments. Alternatively, synthetic tile-based DNA nanotubes are comparable in length and stiffness to cytoskeletal filaments and can be engineered to demonstrate dynamic behavior using few components. To use DNA nanotubes as cytoskeletons in synthetic systems requires resilience to degrading enzymes found within cells, and the dynamic behavior must be automated and characterized in compartments.

Minimal cell systems execute tasks using transcription–translation (TXTL) machinery adapted from native cells. As other DNA nanotechnology degrades rapidly in vivo, I assayed the robustness of DNA nanotubes in an Escherichia coli cell-free TXTL system. TXTL recapitulates physiological conditions as well as strong linear DNA degradation through the RecBCD complex. I demonstrated that chemical modifications of the tiles composing DNA nanotubes and the addition of a Chi-site dsDNA, an inhibitor of the RecBCD complex, extend nanotube viability in TXTL for more than 24 hours. These complementary approaches are a first step towards engineering resilient DNA nanotubes for application in active environments.

To demonstrate autonomous control of assembly and disassembly processes of nanotubes in cell-sized environments, I implemented a DNA-RNA hybrid nanotube design inside of water-in-oil droplets. In this design, DNA tiles are activated by the presence of a trigger RNA molecule, which can be produced by and degraded by distinct enzymes. A pulse of nanotube assembly-disassembly occurs when both transcribing and degrading components are present with inactive tiles in droplets. Notably, the encapsulated system requires lower concentration of gene to trigger assembly than bulk solution. Varying the concentration of gene and degrading enzymes affects both the kinetics of assembly-disassembly and the morphology of the nanotubes. These methods can be employed to develop more complex dynamics and functionalities of DNA nanotubes as synthetic cytoskeletal filaments in minimal cells.

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