Journal: bioRxiv

Article Title: Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell

doi: 10.1101/2024.04.30.591827

Figure Lengend Snippet: a) Schematic illustration of self-replication by a synthetic DNA replicator encoding DNAP and TP from the Փ29 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 Փ29 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. 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).

Article Snippet: Replication-only reactions were performed with a customized PURE solution II minus T7 RNA polymerase (GeneFrontier Corp.).

Techniques: Expressing, Amplification, Modification, Agarose Gel Electrophoresis, Residue, Generated, Concentration Assay