Dack, C.; Li, B.; Newell, C.; Booth, M. J.

Spatial and temporal organisation of membrane-associated components is fundamental to cellular signalling, yet remains difficult to engineer in minimal synthetic systems. In synthetic cells, DNA and RNA nanotechnology offer programmable molecular organisation at membranes, while in vitro transcription (IVT) enables gene expression-driven regulation. However, integrating these systems within cell-like compartments, such as giant unilamellar vesicles (GUVs), remains challenging due to undesirable interactions between transcription machinery and nucleic acid assemblies. Here, we present a modular strategy that couples in situ RNA production to dynamic DNA localisation at GUV synthetic cell membranes. RNA strands, transcribed within GUVs, function as linkers that recruit DNA-conjugated cargo to lipid membranes, enabling programmable spatial organisation. Using this framework, we achieved reversible membrane localisation through toehold-mediated strand displacement and RNase H-mediated degradation. This work establishes a gene expression-driven platform for programmable and dynamic control of membrane-associated components in synthetic cells, providing a foundation for constructing dynamic signalling assemblies and higher-order cellular behaviours.