Abstract
The integration of biological functions into a single operating system is considered a major challenge in the construction of a synthetic cell. We present autocatalytic selection (ACS) of gene functions as a driver for integrating biological modules in vitro. A gene of interest (GOI) is introduced into a minimal DNA self-replicator and the function of the GOI is linked to transcription, translation or DNA replication through a positive feedback loop. As the encoded function eventually promotes DNA self-replication, the gene variants with greater activity are selected. Using different coupling mechanisms, we demonstrate ACS of three functions: transcription, in situ regeneration of dGTP from dGMP to support DNA replication, and β-galactosidase activity. The latter example illustrates how a function that is not directly related to the Central Dogma can be selected. In addition, we show that metabolically active replicators can be enriched from a library of variants generated by random mutagenesis. This work paves the way for ACS-driven Darwinian evolution of virtually any biomolecule in vitro, streamlining the construction of increasingly complex synthetic cells as well as the engineering of biotechnologically relevant enzymes.
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Acknowledgements
We would like to thank Alicia del Prado and Miguel de Vega (Centro de Biología Molecular Severo Ochoa, Madrid) for kindly providing the purified SSB and DSB proteins, Adilya Timmers (TBI, Toulouse) for plasmid pRS316, Sébastien Nouaille (TBI, Toulouse) for plasmid pBADMyc-HIS-lacZ, Thomas Gosselin-Monplaisir (TBI, Toulouse) for E. coli gDNA, and Sara Castaño Cerezo (TBI, Toulouse) for strain CEN.PK2-1C. We are also grateful to Ana María Restrepo Sierra for discussions on DNA replication, and to GeneFrontier for sponsoring our research and for fruitful discussions.
Funding
This work was financially supported by Agence Nationale de la Recherche (ANR-22-CPJ2-0091-01).
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Sierra Heras, L., Danelon, C. Autocatalytic selection of gene functions in synthetic cells. Commun Biol (2026). https://doi.org/10.1038/s42003-026-10372-z
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DOI: https://doi.org/10.1038/s42003-026-10372-z
