Marshall, Z. | bioRxiv Science

bioRxiv2026

Autonomous Homeostatic Synthetic Cells via Self-Gating DNA Nanopores

Homeostasis is a fundamental hallmark of living organisms, arising from the complex interplay between biochemical reactions and regulatory feedback systems. Reconstituting such self-regulating behaviour in minimal synthetic cells enables continuous, persistent operation of biochemical reactions for extended amount of time. In this work, we demonstrate a minimal homeostatic synthetic cell capable of autonomous flux regulation using DNA nanotechnology and bottom-up synthetic biology. Our homeostatic architecture consists of Giant Unilamellar Vesicles (GUVs) equipped with gated DNA nanopores, encapsulated in vitro transcription (IVT) machinery, and an RNA degradation system. We achieve homeostasis under varying external chemical stimuli specifically varying concentrations of rNTPs by implementing a negative feedback loop between rNTP influx and RNA production. In our system, DNA nanopores facilitate the influx of rNTPs from the external environment, driving internal transcription. Crucially, the transcription process generates RNA "blockers" designed to bind and gate the DNA nanopores, thereby attenuating further rNTP influx. Our system is dynamic as encapsulated RNases slowly degrade the RNA blockers, allowing the pores to reopen as blocker concentration goes down. We first characterise the functionality and gating efficiency of the DNA nanopores using both pre-synthesised and in situ produced DNA and RNA blockers. We then demonstrate that rNTP flux through these pores is sufficient to drive IVT within the GUVs. Finally, by integrating these modules, we demonstrate robust homeostasis: the system maintains a steady-state level of RNA production for up to 16 hours. By harnessing the controllability of negative feedback loop, we demonstrate thresholding of the homeostasis level using single-stranded regulator DNA. This work establishes a versatile framework for engineering adaptive and self-sustaining responsive nanomaterials and synthetic cell chassis.

biophysics↗

bioRxiv2025

QuantGUV: Quantifying Encapsulation Efficiency of Small Molecules in GUVs

1Synthetic cells, constructed through the self-assembly of small molecules, are designed to mimic life-like behaviours by encapsulating functional molecules. For such synthetic cells to accurately replicate cellular reactions, it is critical that the concentrations of encapsulated molecules mirror those in living systems, as reaction kinetics and cellular network states are highly sensitive to these concentrations. However, methods for precisely determining encapsulation efficiency in synthetic cells at single cell resolution have been limited. To address this challenge, we developed QuantGUV, a software-driven, image-based analysis method that determines the concentrations of fluorescent molecules encapsulated within giant unilamellar vesicles (GUVs). We used Quant-GUV to measure the encapsulation efficiencies of fluorescent molecules, ranging in size from 0.5 nm to 20 nm. These measurements were conducted on GUVs formed via the water-in-oil emulsion transfer method under various experimental conditions. Using QuantGUV, we have measured the encapsulation efficiencies of three fluorescence molecules, sulforhodamine B, mEGFP and polystyrene bead, in GUVs formed via the water-in-oil emulsion transfer method. The encapsulation efficiencies for polystyrene bead was close to 100% in most of the conditions while sulforhodamine B and mEGFPs encapsulation efficiencies depended on the parameters during the GUV formation such as concentrations of lipids and oil-water ratio during the GUV formation. By providing crucial insights into encapsulation efficiencies, QuantGUV offers a valuable tool to monitor the building quantitative synthetic cell systems with accurately controlled internal environments which is a critical step towards the creation of synthetic cells.

synthetic biology↗