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. 2024 Oct 22;15:9091. doi: 10.1038/s41467-024-53226-0

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic illustration of self-replication by a synthetic DNA replicator encoding DNAP and TP from the Φ29 bacteriophage. Expression in PURE system leads to an autocatalytic network resulting in exponential amplification of the two-gene DNA template. b In-liposome IVTTR of original and modified ori-p2p3 templates assayed by agarose gel electrophoresis of PCR-recovered DNA. c Predicted structures of original T7 leader sequences in ori-p2p3 and an artificial alternative in mod-ori-p2p3. RNAfold webserver drawing of minimum free energy plain structure is shown with indicated base pair probabilities. d Crystal structure of Φ29 DNAP (PDB 2PY5) with the exonuclease domain highlighted in red and F62 residue in blue. The adapted protein structure was generated with PyMOL Molecular Graphics System. e Assay of ori-pssA amplification in bulk IVTTR reaction by DNAP (WT or F62Y) and TP expressed from non-replicating circular template. The concentration of ori-pssA DNA was quantified by qPCR before and after (16 h) IVTTR, showing similar amplification levels for both polymerases. Individual symbols correspond to data from biological replicates (independent experiments). Source data are provided as a Source Data file.