pH-responsive synthetic cells for controlled protein synthesis and release

Abstract

Lipid membrane-bound synthetic cells provide programmable, cell-like compartments for on-demand biomolecule production. However, most cell-free gene expression systems in synthetic cells are regulated by user-imposed cues rather than signals associated with environmental or physiological states. Here, we present an acidity-transducing synthetic cell that converts external pH changes into nucleic acid information to drive in situ protein synthesis. The system integrates gramicidin A proton channels for pH sensing, triplex-forming single-stranded DNA (ssDNA) that releases a trigger ssDNA upon acidification, and a toehold switch RNA that activates translation in response to the released trigger ssDNA. This work further reveals that tuning the annealing length between the pH-responsive and trigger ssDNAs controls the trigger-release pH, which is critical for enabling acid-triggered protein synthesis. The synthetic cells retain their pH-responsive activity when embedded in alginate hydrogels, creating acidity-responsive materials that synthesize proteins in situ rather than release preloaded cargo. Cell-penetrating peptide tagging further enables selective protein release and target-specific binding. This work establishes a molecular transduction strategy for programming synthetic cell materials to sense environmentally relevant acidity and generate functional biomolecules on demand.