Kaletta, N. | Search bioRxiv

bioRxiv2026

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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/728287v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1db026aorg.highwire.dtl.DTLVardef@17bb05dorg.highwire.dtl.DTLVardef@177ca3aorg.highwire.dtl.DTLVardef@17dcf9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

Co-transcriptional Phase Separation of Nucleic Acids at Membrane Surfaces

Transcription is usually framed as information transfer, yet it also injects a new polymer into a crowded, confined environment. Here we demonstrate how spatial confinement to surfaces in a minimal membrane-bound transcription (MBT) system displays the physical consequences of RNA synthesis. Within a dense membrane-tethered DNA network, transcription drives co-transcriptional RNA phase separation: nascent RNA oligomerizes, gels and demixes from a surrounding fluid DNA phase, generating stable spatial patterns while mechanically remodeling the DNA layer. RNA gelation sequesters T7 RNA polymerase, whereas RNA-binding and translation-associated factors reverse gelation and restore fluidity. Thus, in the absence of downstream regulatory machinery, transcription under confinement is sufficient to trigger RNA condensation and nucleic-acid phase separation. The membrane as confining interface catalyzes the onset of DNA-RNA demixing and modulates the morphology of the resulting patterns. Since such large-scale spatial unmixing may be detrimental to cellular physiology, we suggest that one fundamental role of translation is to actively prevent condensation effects created by continuous RNA production.

biochemistry↗