PURErep enables self-encoded DNA replication

Initially, we tested self-encoded Phi29-DNAP-dependent TTcDR using the standard protocol of the commercially available PURExpress system. The Phi29-DNAP coding region flanked by a T7 promoter was first cloned into a pCR-Blunt TOPO vector (pREP, Fig. 1a). In principle, this construct should enable spontaneous RNA-primed rolling-circle replication13 by the self-encoded DNAP without additional replication proteins or externally supplied DNA primers as reported previously10. However, using the standard PURExpress reaction supplied with dNTPs and 4 nM pREP, we were unable to detect de novo synthesis of DNA by either agarose gel electrophoresis or qPCR (Fig. 1b, c). This finding is in agreement with previous studies which reported that the high tRNA and rNTP concentrations in standard PURE systems impair DNA-polymerase (DNAP) activity and that optimised custom systems are required to achieve efficient TTcDR10,11. In order to improve DNA replication without access to tailor-made PURE systems, we set out to optimise the PURExpress standard reaction protocol. To this end, we increased the relative amount of translation factors, ribosomes and reducing agent while decreasing tRNA and rNTP levels (Fig. 1d; Supplementary Table 1). Using this optimised PURE formulation (PURErep), we achieved, depending on the pREP input concentration, ∼5–12-fold replication of pREP monomer units in overnight TTcDR reactions (Fig. 1b, e). Full-length de novo synthesis of pREP was confirmed by MluI digestion of the replication product (Fig. 1c). Taking superfolder green fluorescent protein (sfGFP)14 expression as an overall measure for in vitro translation (IVT) activity, we found that the altered PURE formulation resulted in a batch-dependent reduction of protein synthesis yields of 20–40% compared with the TTcDR-incompetent PURExpress system (Supplementary Fig. 1A, B). Thus, the improved compatibility of the PURErep system with DNA replication is achieved at the expense of only a modest reduction in overall protein expression strength.

Fig. 1: PURErep enables efficient transcription–translation coupled DNA replication. a Map of pREP. The plasmid encodes the gene for Phi29-DNAP under the control of a T7 promotor (T7p) and a bidirectional T7-terminator (T7t). IVT of the DNAP gene is increased by a T7 g10-L leader sequence. A zeocin-resistance gene and a pUC origin allow selective propagation in E. coli. b Replication of pREP in PURExpress (grey) or PURErep (blue) after 6 h at 30 °C. Fold changes were determined by qPCR relative to the pREP input levels (4 nM). Bars show the means with their 68% confidence intervals (CI) from biological triplicates using different PURErep/PURExpress batches. c Image of representative agarose gels loaded with TTcDR samples of pREP (8 nM input DNA) after MluI treatment. Samples were tested in three biological replicates. d Relative changes in compound levels between PURErep and PURExpress (log 2 -scale). Estimated compound levels for PURExpress are based on the numbers from Kuruma and Ueda40 (TF translation factors, AAs amino acids, DTT dithiothreitol, 10-THF 10-Formyltetrahydrofolate, SP spermidine, CP creatine phosphate). e TTcDR of pREP at different input concentrations. Fold changes relative to input levels were measured by qPCR (means with 68% CI, biological triplicates using different PURErep batches for each concentration). f pREP propagation over repeated passages of serial transfer. After each overnight TTcDR reaction in PURErep, 4% of the volume was transferred into a freshly prepared, plasmid-free PURErep reaction. Fold changes relative to the initial concentration (4 nM) were used to approximate the concentrations before and after each generation (mean ± 68% CI, technical triplicates). Source data are available in the Source Data file. Full size image

TTcDR products can be transformed and propagated in E. coli

A qPCR-based analysis of DNA replication revealed a robust doubling time of 1–2 h for different initial template concentrations with DNA replication proceeding even after 24 h at 30 °C (Fig. 1e). TTcDR of pREP was also sustainable for more than five successive generations of serial dilution when 4% of an overnight PURErep/pREP reaction was directly transferred into a fresh PURErep mix (Fig. 1f). This result implies that TTcDR products can serve as templates for self-coded DNA replication over several generations. As expected from the rolling-circle-type replication, we observed a considerable amount of product with low electrophoretic mobility, likely representing large molecular weight concatemers and/or DNA-MgPP i clusters as reported previously for similar reactions (Supplementary Fig. 1C)15. Unexpectedly, we also observed formation of ~5 kb products in unprocessed samples, suggesting that TTcDR reactions may produce considerable amounts of monomeric pREP copies (Supplementary Fig. 1C). We were also able to transform de novo synthesised products into E. coli after removal of parental plasmids (Supplementary Fig. 2A). Purified in vivo amplified products were identical in size to monomeric pREP (Supplementary Fig. 2B).

PURErep enables TTcDR of large multipartite genomes

Encouraged by the efficient TTcDR in PURErep, we set out to co-replicate a collection of genes coding for crucial components of the PURE reaction such as the 31 essential E. coli translation factors (TFs). To this end, we probed co-TTcDR of pREP (4.6 kb) together with each one of the three large plasmids pLD1 (30 kb, 13 translation factors – TFs), pLD2 (20 kB, 8 TFs), or pLD3 (23 kb, 9 TFs), which were recently cloned to enable recombinant expression of 30 of the 31 TFs16. Indeed, the TTcDR products of all four plasmids (including pREP) showed identical MluI restriction patterns as clonal plasmids conventionally propagated in E. coli (Fig. 2a). Moreover, the pLD TTcDR products could be directly transformed into E. coli, from where they were maintained as monomeric plasmids (demonstrated for pLD3, Supplementary Fig. 2C, D). The optimised PURErep mix enabled even the complete replication of all three pLD plasmids together with PURErep in a one-pot reaction (Fig. 2b; Supplementary Fig. 3A, B).

Fig. 2: In vitro replication of large multicistronic DNA constructs. a MluI restriction patterns of gel-purified TTcDR products from individual pLD/pREP co-replication experiments at t = 0 h and t = 16 h. Concentrations were 6 nM pREP and 0.7 nM pLD1, pLD2 or pLD3. Authentic control standards for clonal pREP/pLD mixtures are shown for each TTcDR reaction. The raw gel image is shown in Supplementary Fig. 3A. Samples were tested in three biological replicates. b Representative restriction digest of individual pLD co-replication experiments (lanes 1–3, 2 nM pLD plasmid, 4 nM pREP) and co-replication of all pLD plasmids (lane 4, 4 nM pREP, 2 nM pLD1-3). The specific MluI cleavage products for each plasmid are colour-labelled (pREP—cyan, pLD1—green, pLD2—red, and pLD3—purple). To improve the visibility of low-molecular-weight bands, the lower parts of the bands are presented with different contrast settings (indicated by the dotted line). The unprocessed gel images are shown in Supplementary Fig. 3B. Samples were tested in biological replicates. c Fold changes of the six plasmids pREP, pLD1-3, prRNA and pEFTu after an overnight TTcDR relative to their respective input concentrations determined by qPCR using plasmid-specific amplicons (shown are means with their 68% CI from biological triplicates using different PURErep batches). d LB plates of E. coli 10-beta cells transformed with overnight TTcDR reactions from c. TTcDR reactions were carried out in presence ( + dNTPs) or, as negative control for background colonies, in absence (-dNTPs) of dNTPs. Cell-derived plasmid templates were digested with DpnI. Cells were grown under selective conditions for zeocin (Zeo, pREP), kanamycin (Kan, pLD1-3, prRNA) and carbenicillin (Cb, pEFTu). e Circle diagrams of the relative transformation frequencies for the six plasmid species isolated from 26 randomly picked colonies from the three +dNTP plates in d (7 for Zeo, 12 for Kan and 7 for Cb). For the restriction pattern analyses, see Supplementary Fig. 3C–E. Source data are available in the Source Data file. Full size image

Next we sought to further expand the genetic load of the TTcDR-system by co-replicating plasmids encoding additional components of the PURE system such as EF-Tu (pEFTu), which is missing in the pLD system, and also the ribosomal RNA operon rrnB (prRNA), which encodes for 23S rRNA, 16S rRNA, 6S rRNA and tRNA(Glu2)17 (Fig. 2c). qPCR experiments targeting plasmid-specific amplicons confirmed that monomer units of all six plasmids (total DNA length 93 kb) were replicated about 2–8-fold relative to their respective input levels in the presence of pREP and dNTPs after overnight incubation (Fig. 2c). In support of complete co-replication of all plasmids, transformations of DpnI-treated PURErep reaction products into E. coli resulted in colonies resistant to either zeocin (pREP), kanamycin (pLD plasmids and prRNA) or carbenicillin (pEFTu) (Fig. 2d). DNA preparations of 26 randomly picked clonal colonies followed by restriction pattern analysis indeed confirmed successful TTcDR of all six plasmids (Fig. 2e; Supplementary Fig. 3C–E). In contrast, almost no background colonies were obtained when samples from dNTP-free PURErep experiments were transformed into E. coli (Fig. 2d). Using the same approach (Fig. 3; Supplementary Fig. 4), we were able to demonstrate co-replication of five additional plasmids encoding all but one of the missing proteins of the PURE enzyme mix (Supplementary Table 2, except peptidylprolyl isomerase). The additional plasmids include the genes for a minimal nucleoside triphosphate regeneration system based on creatine kinase (pCKM), adenylate kinase (pAK1) and nucleoside diphosphate kinase (pNDK), as well as T7-RNA polymerase (T7RNAP) and pyrophosphatase (pIPP), which is added to more recent versions of the PURE system18. With a total size of 116.3 kb, this set of 11 plasmids reaches >100% of the predicted genome length proposed for a near-minimal, self-replicating system dependent only on small-molecule nutrients (Fig. 3a)3.

Fig. 3: In vitro replication of a 116.3 kb multipartite DNA genome. a The collection of 11 plasmids (combined length 116.3 kb), which were co-replicated in PURErep. In addition to Phi29-DNAP (pREP, 6 nM input), the plasmid ensemble encodes the ribosomal rRNA operon rrnB (prRNA, 3 nM input), all E. coli TFs (pLD1-3, pEFTu, 0.6 nM input each), a minimal NTP regeneration system (pNDK, pCKM, pAK1, 3 nM input each), T7-RNA polymerase (pT7pol, 3 nM input) and inorganic pyrophosphatase (pIPP, 3 nM input). b LB plates of E. coli 10-beta cells transformed with overnight TTcDR reactions containing all 11 plasmids from a. TTcDR reactions were carried out in presence (+dNTPs) or, as negative controls, in absence (−dNTPs) of dNTPs. Cell-derived plasmid templates were digested with DpnI after TTcDR. Cells were grown under selective conditions for zeocin (Zeo, pREP), carbenicillin (Cb, pAK1, pIPP, pEFTu), chloramphenicol (Cm, pNDK, pT7RNAP, pCMK) and kanamycin (Kan, pLD1-3, prRNA). c Circle diagrams of the relative transformation frequencies of the 11 plasmid species isolated from 41 randomly picked colonies from the four + dNTP plates in B (5 for Zeo, 12 for Cb, 12 for Cm and 12 for Kan). For the restriction pattern analyses, see Supplementary Fig. 4. Source data are available in the Source Data file. Full size image

PURErep enables synthesis of 30 TFs during TTcDR

Having shown combined TTcDR of the multicistronic plasmids that encode almost all proteins of the PURE enzyme mix, we explored whether the PURErep mix could also enable parallel expression of these genes during replication. A (partially) self-replicating system based on the central dogma needs to be able to regenerate at least some of its different protein components. As a first step in this direction, we focused on the multicistronic expression of the TFs encoded on the three pLD plasmids pLD1, pLD2 and plD3 (not including pEFTu). To explore whether PURErep is generally capable of supporting multicistronic expression from these plasmids, we performed cell-free expression from each individual plasmid in presence of BODIPY-Lys-tRNA Lys , which enables the fluorescent labelling of translation products at lysine residue sites. Using the reported expression patterns for affinity-purified TF ensembles from pLD overexpression experiments16, we could assign the majority of the de novo synthesised protein subunits to the to the respective TFs (Supplementary Fig. 5). To improve detection sensitivity and enable quantification of newly synthesised proteins, we also performed a mass spectrometry-based quantitative protein expression analysis using stable-isotope labelling19. For this purpose, we carried out PURErep in vitro experiments with each pLD plasmid with 15N 2 13C 6 -lysine as sole source of lysine and 15N 4 13C 6 -arginine as the sole source of arginine. Using the unlabelled PURE-supplemented TFs as internal standards to determine the heavy-to-light (H/L) ratio of isotope-labelled peptides, we found strong evidence for the de novo synthesis of all pLD-encoded TF protein subunits in overnight reactions (Fig. 4a). In particular, we obtained H/L ratios close to or larger than one for 12 of the 13 TFs encoded on pLD1 implying full regeneration of most of the encoded proteins during IVT. Partial or even full regeneration was also observed for the proteins encoded on both pLD2 and pLD3 (Fig. 4a).

Fig. 4: A substantial number of TFs are efficiently expressed during co-replication of pLD1, plD2, pLD3 and pREP. a H/L ratios of the 32 TF protein subunits after 15N 2 13C 6 -lysine and 15N 4 13C 6 -arginine incorporation during in vitro transcription/translation of pLD1 (green, n = 4), pLD2 (red, n = 3) or pLD3 (purple, n = 4) in PURErep overnight reactions. The line H/L = 1 indicates full regeneration of a protein to its respective input concentration. b Expression levels during parallel TTcDR of pLD1 (green), pLD2 (red) and pLD3 (purple) during TTcDR induced by the addition of pREP. All H/L values are means + /− s.d. (n = 3) from biological replicates triplicates using different PURErep batches. Source data are available in the Source Data file. Full size image

Next, we probed multicistronic expression of all three pLD plasmids during parallel TTcDR induced by the addition of pREP. Despite the considerably increased synthetic burden (replication of a 78 kb genome and transcription/translation of 33 protein chains), we detected H/L ratios > 0.73 for 16 of the 32 encoded protein subunits. The H/L ratios of remaining TF subunits indicated regeneration levels between 10–70% (N = 10) and 4–9% (N = 6) (Fig. 3b). Thus, even under non-optimised batch conditions, PURErep in combination with pREP enables both the complete replication of 32 pLD-encoded TF cistrons as well as expression of about half of the encoded TF peptide chains in yields comparable or exceeding their initial PURErep input concentrations.