Developing Resilient DNA Polymers for Operation in an E. coli Transcription-Translation System

DNA nanotechnology is a growing field with potential intracellular applications. Interfacing DNA nanostructures with biology, however, faces many challenges, a major one being the resilience of such devices in vivo. In this work, we use an Escherichia coli cell-free transcription–translation (TXTL) system to assay the robustness of DNA nanotubes in a cytoplasmic environment. TXTL recapitulates physiological conditions as well as strong linear DNA degradation through the RecBCD complex, the major exonuclease in E. coli. We demonstrate that chemical modifications of the tiles making up DNA nanotubes extend their viability in TXTL for more than 24 hours, with phosphorothioation of the sticky end backbone being the most effective. Furthermore, we show that a Chi-site dsDNA, an inhibitor of the RecBCD complex, extends DNA nanotube lifetime significantly. These complementary approaches are a first step towards a systematic prototyping of DNA nanostructures in active cell-free cytoplasmic environments and expand the scope of TXTL utilization for bioengineering.