DNA condensate-based organelles for spatially regulated gene expression and protein targeting in synthetic cells

Spatial organization of gene expression is a key feature of cells but remains a major challenge in bottom-up synthetic biology. While phase-separated DNA condensates have been used to spatially confine transcription, achieving efficient recruitment of protein-coding DNA for full protein biosynthesis within these membrane-less structures has remained a major challenge. Here, we present a modular DNA nanostructure that enables tunable and highly efficient partitioning of long client DNA into condensate-based synthetic nuclei, thereby surpassing present limitations. This serves as the starting point for a multimodal DNA organelle-based system for spatially regulated gene expression in synthetic cell environments. The flow of information includes localized transcription within this synthetic nucleus, followed by translation and product release into the surrounding cytosol. Furthermore, we extend the toolkit of spatiotemporal organization by designing protein targeting of cell-cycle protein ParR within parC-enriched orthogonal DNA condensates. Quantitative analysis reveals a trend toward higher protein yield in the condensate-based system compared to standard PURE expression, indicating a functional advantage of spatial organization even in such minimal systems. Hence, we demonstrate how protein expression can be engineered not only by molecular composition, but by spatial architecture itself.