Bootstrapping DNA replication with ribonucleotide reductase in a minimal cell-free system

Bootstrapping DNA replication with ribonucleotide reductase in a minimal cell-free system | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bootstrapping DNA replication with ribonucleotide reductase in a minimal cell-free system Hannes Mutschler, Jacopo De Capitani, Noemi Nwosu, Viktoria Gocke, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9248024/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract All living organisms must maintain a balanced pool of DNA building blocks to replicate and propagate their genetic information. In living cells, ribonucleotide reductase (RNR) is the central enzyme responsible for dNTP synthesis. In contrast, minimal protein-based cell-free systems, are powerful platforms for reconstituting biological processes in vitro, but lack the endogenous metabolic pathways required to autonomously replicate their DNA and rely entirely on externally supplied dNTPs. This dependency prevents cell-free systems from achieving the metabolic autonomy required for self-sufficient genetic replication. In this work, we successfully integrated RNR’s redox activity and complex allosteric regulation for in situ synthesis of all dNTPs from endogenous NTP pools. We combined RNR’s activity to DNA synthesis and propagation of the genetic information encoding a self-contained minimal DNA replication machinery. The replication products retain the genetic information and enable re-booting of self-encoded RNR-dependent DNA synthesis. Using this strategy, cell-free systems with self-sufficient dNTP metabolism may open new avenues toward completely autonomous synthetic cells. Biological sciences/Systems biology/Synthetic biology Biological sciences/Biochemistry/DNA Biological sciences/Molecular biology/DNA replication/DNA synthesis Biological sciences/Molecular biology/DNA metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Artificial minimal protein-based systems allow the study of complex dynamic biological processes in environments with controlled compositions. 1‑6 The PURE (Protein Synthesis Using Recombinant Elements) in vitro transcription-translation system has served as a minimal backbone to explore key reactions associated with the central dogma. 7,8 Notably, PURE can be augmented to display life-like processes outside of a cellular context, including self-regeneration of the translation machinery 9‑18 and replication 19‑22 and evolution 23‑27 of genetic information. Enabling these functions requires increased metabolic complexity within the standard PURE system. 28,29 Recent efforts have successfully integrated pathways for in situ synthesis of amino acids 30,31 and energy regeneration. 32‑34 However, the absence of metabolic complexity surrounding DNA replication represents a critical bottleneck that prevents the development of truly “life-like” minimal cell systems. Since PURE is an extremely heterotrophic multi-enzyme system, replication of its genetic content has remained dependent on the external supply of additional nucleobases or on the partial regeneration of nucleic acid building blocks through limited re-phosphorylation. 35‑37 In this work, we sought to augment PURE to enable the in situ synthesis of DNA building blocks and thereby reduce PURE’s dependency on external supply of DNA building blocks (Supplementary Fig. 1). We integrated the expression of E. coli ribonucleotide reductase (RNR) Ia to synthesize dNTPs from available ribonucleotide pools, generated as part of the energy regeneration machinery in PURE. We verified the reliability of this system and confirmed that RNR Ia could efficiently supply all dNTPs for DNA synthesis, by coupling RNR Ia’s multi-turnover activity to the transcription of a fluorescent light-up RNA aptamer. 38 The synthesized dNTPs could be used to drive the replication of the plasmids encoding for a self-contained minimal DNA synthesis and replication metabolic network, encompassing the self-encoded RNR Ia and Phi29 DNA polymerase (DNAP). Results Characterization of RNR Ia activity in PURE using P-FLARE assay. The 261 kDa RNR Ia heterodimer tetrameric complex 39 can promiscuously reduce any nucleoside 5’-diphosphate (NDP) into its deoxynucleoside 5’-diphosphate (dNDP) form, through radical-based redox catalysis 40,41 and complex allosteric regulatory mechanisms of substrate and effector binding, 42,43 (Fig. 1a). Synthesis of the individual dNDPs by RNR Ia requires a sophisticated long-range, reversible Proton-Coupled Electron Transfer (PCET) as well as a complex orchestration of a series of intramolecular dynamics and efficient communication between the a2 and b2 homodimers. Upon coordination of Fe 2+ and formation of the Fe 3+ -Y 122 · radical in the b subunits (Figure 1a), 41 the PCET between the b and a subunits 44,45 enables the generation the thyil radicals in the a subunit active site, 46 with which the assembled a2b2 complexes can reduce any NDP. The regulatory mechanism and multi-turnover reduction activity of RNR Ia in vitro depends on the presence of several cofactors, such as sufficient free Mg 2+ to facilitate intermolecular interactions between the a2 and b2 homodimers, 47 enough ATP to ensure the formation of the a2b2 active complex 48,49 and the optimal concentration of TCEP reducing agent to assist the regeneration of the catalytic cysteines in the a subunit. 50 The activity of RNR Ia could potentially be seamlessly integrated within the existing pathways of the PURE system (Fig. 1b), where NDPs are generated as part of aminoacylation, translation and regeneration of energy currencies. De novo synthesized RNR Ia could then use these NDPs to provide substrates to DNA synthesis by Phi29 DNA polymerase (DNAP). To probe if the reaction environment of the PURE system is compatible with these intricate enzymatic mechanisms and if it can sustain the multi-turnover reduction of different NDP substrates, we adapted the Fluorescent Light-up Aptamer RNR Enzymatic assay (FLARE) 51 for detection in PURE (i.e. P-FLARE) (Fig. 2a). The read-out of the FLARE assay relies on the reduction of NDPs by RNR Ia; the nucleoside diphosphate kinase (NDK) already present in PURE, phosphorylates the dNDPs generated by RNR Ia into dNTPs which can then be used by Phi29 DNAP to extend a partially double stranded DNA (dsDNA) template of the Broccoli fluorescent light-up aptamer (FLAP). Completion of this dsDNA template establishes a functional T7 promoter (T7p), enabling T7 RNA polymerase (RNAP) to transcribe the Broccoli FLAP, resulting in detectable green fluorescence upon binding of the fluorogen DFHBI-1T. Using P-FLARE, we confirmed that recombinant RNR Ia supplied to PURE could generate all sufficient dNDPs to generate a detectable signal (Supplementary Fig. 2). We then verified that expressed RNR Ia could assemble in its active a2b2 complex from subunits expressed in similar rations from equimolar input plasmids (Fig. 2b and Supplementary Fig. 3). To test the RNR-dependent reduction of NDPs in PURE, we first adopted a dCTP-limiting set-up in P-FLARE (i.e. we supplied CDP and dDTPs – dATP, dGTP, dTTP) to confirm that either one (Supplementary Fig. 4) or both de novo synthesized RNR Ia subunits (Fig. 2c) could generate the dCTP necessary for DNA synthesis. When either subunit was expressed alongside the other supplied purified subunit, the resulting a2b2 complex reduces the CDP supplemented to PURE and helps generate the dCTP required for dsDNA synthesis and subsequent Broccoli FLAP transcription (Fig. 2d). However, when only the a2 homodimer was expressed to form the complex with supplied purified b2, this resulted in a lower maximum fluorescence signal (69% ± 6% of RNR-independent P-FLARE), likely due to suboptimal subunit ratio between expressed a2 and supplied b2. 48 When both subunits are expressed simultaneously in roughly equimolar ratios, the fluorescent signal reached similar intensities as in RNR-independent reactions, while incurring in a signal delay due to the necessary expression of both subunits (Fig. 2d). The observed P-FLARE rates are comparable irrespective of which subunit is expressed, which suggested that the formation of the active RNR Ia complex was the rate limiting step for the assay’s read-out (Fig. 2e). Using this dCTP-limiting set-up, we crucially confirmed that the observed fluorescent signals depended on de novoRNR Ia correctly generating the Fe 3+ -Y 122 · radical (Supplementary Fig. 5) and establishing a functional PCET between the two homodimers (Supplementary Fig. 6). Establishing deoxynucleotide metabolism in PURE using RNR Ia. We then investigated the synthesis of dNTPs from NDPs endogenously generated in PURE. The dephosphorylation of NTPs by NDK during energy regeneration produces enough NDPs to serve as a substrate for RNR Ia for the reduction into dNDPs. These are subsequently re-phosphorylated by NDK into dNTPs that can be utilized by Phi29 DNAP to synthesize new DNA. By testing different input concentrations of CDP in a dCTP-limiting P-FLARE assay, we observed that P-FLARE rates were consistently independent of exogenously supplied CDP (Supplementary Fig. 7). Using P-FLARE reactions with specific dNTP-limiting conditions (e.g. when ADP had to be reduced, we added only dBTPs – dGTP, dCTP, dTTP), we confirmed that each of the four NDPs endogenously generated in PURE could be reduced by RNR Ia at similar rates to when the same NDP was supplemented to the reactions. (Fig. 3a and Supplementary Fig. 8). Encouragingly, P-FLARE reactions displayed similar rates when read-out depended on the reduction of either ADP or GDP, with a slight decrease when it depended on CDP reduction. In contrast, reactions that required the synthesis dUTP and, subsequently, uracil-DNA (u-DNA) for read-out, showed starkly diminished read-out signals (Fig. 3b and Supplementary Fig. 8c). This decrease in RNA synthesis could be a result of lower incorporation rates of dUTP by Phi29 DNAP or reduced dUTP synthesis rates by RNR Ia. This effect was to be expected given that in living cells dUTP is usually converted into dTTP by thymidylate synthase and thymidylate kinase. 52‑54 To disentangle these potentially compounding effects, we tested P-FLARE read-out hinging on the synthesis of u-DNA in the presence of supplemented dUTP (Supplementary Fig. 9). In this set-up we observed lower rates and Broccoli yields when dUTP was supplemented instead of dTTP in RNR-independent P-FLARE read-outs, which can be explained by lower incorporation rates of dUTP by Phi29 DNAP, as previously observed. 51 Instead, when P-FLARE read-out depended on the activity of both Phi29 DNAP and RNR Ia, lower concentrations of dUTP synthesized by RNR Ia resulted in a further decrease in RNA synthesis. As such, the coupled activity of RNR Ia and Phi29 DNAP guaranteed that when no dNTPs or NDPs were supplied to PURE, RNR Ia could supply all necessary dNTPs for DNA synthesis, despite a compounding metabolic effect (Fig. 3b, Supplementary Fig. 8c and Supplementary Fig. 9). The fact that newly synthesizedRNR Ia could convert all endogenous NDPs into their respective dNDPs was a crucial step for the development of a self-contained DNA synthesis metabolic network. This observation suggested that de novoRNR Ia maintained the allosteric regulation in which specific reduced dNTPs function as the effectors for the reduction of subsequent NDPs, following a “reduction cascade”. 43 Using P-FLARE in PURE, we mimicked the intracellular regulation of de novo DNA building by simulating this precise regulatory pattern observed in mechanistic studies 42,43 (Supplementary Fig. 10). We probed RNR Ia reduction of various endogenous NDPs using all 16 possible input dNTP combinations (Supplementary Fig. 11). Reaction rates were generally consistent irrespective of which NDP combination had to be reduced, apart from when dUTP had to be synthesized from available UDP, which resulted in lower downstream transcription of Broccoli FLAP. As we observed previously, 51 RNR Ia relies on available dTTP as the specificity effector for dGTP generation. Nonetheless, in the absence of dTTP, RNR Ia notably appeared to be capable of using dUTP as a weak-specificity effector to initiate GDP reduction instead of dTTP. This is evidenced by the detection of reduced but highly significant (p=0.003) P-FLARE activity in all samples where both dGTP and dTTP were omitted (see Supplementary Fig. 11). Therefore, similarly as in deoxynucleotide de novobiosynthetic pathways in E. coli, 55‑57 in PURE, RNA building blocks (i.e. NTPs) are de-phosphorylated by NDK into NDPs, which can serve as substrate for RNR Ia for in situsynthesis of all dNTPs, which can subsequently be incorporated into nascent DNA. Autocatalytic RNR-dependent DNA replication of plasmids encoding RNR Ia. After confirming that all dNTPs could be synthesized in situin PURE by de novo expressed RNR Ia, we coupled RNR Ia activity to the replication of its encoding pUC-a and pUC-b plasmids (Supplementary Fig. 12). We designed this metabolic pathway so that Phi29 DNAP could rely solely on dNTPs generated by self-encodedRNR Ia starting only from endogenous NDPs. With this system, we therefore coupled the metabolic activity of RNR Ia to the autocatalytic propagation of its own genetic information. Rolling-circle amplification (RCA) of the pUC RNR vectors relied on RNA primers being generated by the exonuclease activity of Phi29 DNAP, therefore reducing the heterotrophic dependency on external input of additional Phi29-related proteins or exonuclease-resistant DNA primers. 19,20,58 We first optimized a composition of PURE to couple the expression of RNR Ia with whole-plasmid replication (i.e. transcription-translation-coupled DNA replication – TTcDR PURE). 19,21,59 After adjusting the formulation of TTcDR PURE to accommodate the required reducing agent for RNR Ia 50 (Supplementary Fig. 13), we observed that TTcDR PURE was less sensitive than PURExpress when used for the P-FLARE assay (Supplementary Fig. 14), but it substantially improved replication of pUC RNR vectors compared to PURExpress (Supplementary Fig. 15). Using this optimized TTcDR PURE composition, we confirmed strictly RNR-dependent TTcDR of the input plasmids by transforming the DNA resulting from TTcDR in E. coli cells (Fig. 4a, 4b). Replication of both pUC-a and pUC-b plasmids depended on the synthesis of either dCTP (i.e. supplying dDTPs), dVTPs (i.e. supplying only dTTP), or all dNTPs by de novoRNR Ia. The transformation of the products of DNA replication resulted in ~10 4 colony-forming units per mL (CFUs/mL), when both pUC-a and pUC-b plasmids were supplied. In contrast, the number of background CFUs/mL was reduced to less than 10 2 when either pUC-a or Phi29 DNAP were omitted from the reactions. (Fig. 4b, 4c). As such, this semi-quantitative read-out of plasmid replication, demonstrated that the efficiency of RNR-dependent TTcDR did not hinge on the number of unique dNTPs that had to be synthesized by RNR Ia. We further confirmed that the transformed and propagated TTcDR products contained full-length plasmid sequences using restriction mapping and Sanger sequencing of isolated of pUC-b plasmid DNA from sampled colonies (Fig. 4d and Supplemental Data). To further validate the replication of the genetic information encoding for RNR Ia, we adapted the P-FLARE assay to rely on the expression of functional RNR Ia starting from the DNA generated during TTcDR (Fig. 4e). Specifically, the expression of RNR Ia, and hence the P-FLARE read-out, depended on the amounts of RNR Ia-encoding DNA produced by the upstream TTcDR reaction, which on the other hand was dependent on the reduction of either dCTP, dVTPs or all dNTPs. After extensive DpnI incubation to remove the parental pUC-a and pUC-b plasmids, a fraction of the TTcDR reaction was used as input for P-FLARE reactions in PURE (Fig. 4e). We anticipated that increased DNA replication yields in TTcDR would result in higher expression yields of RNR Ia in P-FLARE, which in turn would result in increased amounts of dsDNA template generated by Phi29 DNAP and therefore enhanced transcription yields of Broccoli FLAP by T7 RNAP. We recorded a significant increase of Broccoli fluorescence in P-FLARE reactions supplied with the DNA products from TTcDR reactions dependent on dCTP synthesis by the self-encoded RNR Ia. Here, fluorescence readouts reached 35 % ± 3% of the P-FLARE reactions supplied with the products of the RNR-independent TTcDR (i.e. supplied with all four dNTPs) (Fig. 4e). In contrast, when the upstream TTcDR reactions relied on the synthesis of dVTPs or all dNTPs, we detected only background-level signals. Presumably, the amount of DNA produced in these TTcDR reactions, when diluted in a new P-FLARE reaction, was not sufficient to generate enough functional RNR Ia to kickstart Broccoli transcription in P-FLARE within the lifespan of a bulk transcription-translation reaction in PURE. However, this crucially confirmed that while RNR Ia was expressed at sufficient levels to generate the necessary dNTPs for plasmid replication in TTcDR, the carry-over of expressed RNR Ia and synthesized dNTPs were insufficient to elicit a signal in the downstream P-FLARE reaction. Co-expression of RNR Ia and Phi29 DNAP enables autocatalytic synthesis and replication of minimal DNA replication machinery. Finally, we assessed whether RNR-dependent DNA replication could be introduced in PURE to recreate a more life-like replication of DNA, by mimicking intracellular DNA synthesis where dNTPs are generated by de novobiosynthetic pathways and used for replication of the genetic information encoding for those same pathways. We approached this challenge by designing a system where the activity of a minimal DNA replication machinery, comprising both self-encoded RNR Ia and Phi29 DNAP, drive the replication and propagation of the 13 kbp minimal genome encompassing the metabolic pathway. Using P-FLARE, we first confirmed that when co-expressing both proteins, de novoPhi29 DNAP could efficiently incorporate all dNTPs generated by RNR Ia (Fig. 5a, 5b), and that observed rates where comparable if Phi29 DNAP was either supplied or expressed from the pREP plasmid (Supplementary Fig. 16). Encouragingly, P-FLARE reactions generated detectable signals even when RNR Ia was required to synthesize an increasing number of dNTPs, although reaction rates gradually declined, likely due to reduced translation yields of both proteins and an increased metabolic burden on RNR Ia (Fig. 5b and Supplementary Fig. 17). Using the same reaction set-ups lacking specific or all dNTPs (Fig. 5a), we confirmed that this minimal self-encoded DNA building pathway expressed in PURE could generate all dNTPs in situand utilize them to replicate all the plasmids encoding for this pathway (Fig. 5c and Supplementary Fig. 18). The replication products of all three input plasmids encoding the minimal autocatalytic TTcDR system (pUC-a, pUC-b and pREP) retained their ability for in vivo propagation since transformation of the products of DNA replication resulted in ~ 10 4 to 10 5 CFUs/mL when using selection markers for either the pUC RNR plasmids or pREP (Fig. 5d, 5e). As anticipated, the efficiency of TTcDR was not limited by how many dNTPs had to be synthesized or by the source of Phi29 DNAP, but rather by the activity of expressed RNR Ia (Supplementary Fig. 19). We also confirmed that the transformed and propagated TTcDR products contained full-length plasmid sequences of all three plasmids using restriction mapping and Sanger sequencing of isolated of pUC-b plasmid DNA from sampled colonies (Fig. 5f, 5g and Supplemental Data). In contrast, no background colonies were observed when either pUC-a or pREP were omitted from the reactions. Encouragingly, by kickstarting P-FLARE reactions with DNA replicated with our minimal self-contained DNA synthesis pathway, we confirmed that the concentrations of plasmids replicated during TTcDR followed a similar pattern when Phi29 DNAP was either supplied or co-expressed (Fig. 4a and 5h respectively). When the pUC RNR Ia and pREP vectors, replicated by de novoexpressed Phi29 DNAP, were used to reboot the synthesis of both proteins in P-FLARE, the generated amounts of replicated plasmids could sustain the synthesis of DNA in a dCTP-limiting set-up, reaching 34% ± 5% of the RNR-independent reactions (Fig. 5h). Therefore, the in situsynthesis of dNTPs by RNR Ia created a rate-limiting metabolic step for DNA replication, akin to what is observed in vivoin E. coli cells as part of metabolic pathwaysfor de novodNTP biosynthesis. 55‑57 Discussion In this work, we set out to expand the metabolic capabilities of PURE to advance the development of increasingly autonomous artificial systems able to produce components required for their own self-regeneration and propagation. 60 Starting only from the ribonucleotide pools available in PURE, we reconstructed a de novo dNTP biosynthetic metabolic pathway driven by the expression of RNR Ia. We then integrated RNR Ia activity into a self-contained, autocatalytic DNA synthesis pathway together with Phi29 DNAP, enabling faithful replication of its own genetic and re-transformation and propagation in E. coli cells. The integration of RNR Ia into PURE therefore represented an important step towards the development of a metabolically self-sufficient TTcDR system. Such a system would rely on its own components to replicate its own DNA while using the allosteric regulation of RNR Ia to dynamically tune dNTP production for continued propagation. 5,28 Several challenges remain to improve the self-sufficiency and self-regenerative capabilities of the system. Competition of limited resources between transcription, translation and DNA replication likely reduces RNR Ia yields, 59 limits the availability of NDP substrates, and decreased DNA replication rates. As a result, compared with reactions where all dNTPs were supplied exogenously, RNR-dependent reactions showed a 30-40% decrease in reaction rates in P-FLARE reactions and about 35% fewer colonies observed during plasmid in vivo propagation. These effects are likely the result of mismatch between the expressed RNR Ia and the regeneration of its required cofactors (i.e. ATP regeneration and free Mg 2+ ) in PURE, or sub-optimal stoichiometries between the expressed RNR Ia subunits, both of which could in turns lead to unfavorable ratios of active and inactive RNR Ia complexes. 48,49 Addressing these constraints will likely require optimization of the expression levels of the RNR Ia subunits and of Phi29 DNAP to improve dNTP and DNA synthesis. In addition, incorporating a dTTP synthesis pathway into PURE will help to overcome the current limitation of u-DNA synthesis. Including thymidylate synthase in PURE will further improve the allosteric regulation RNR Ia and the efficiency of nucleotide incorporation rate by Phi29 DNAP relative to dUTP. Alternatively, the integration of dNTP metabolism in PURE may benefit from the use of RNR Ia variants optimized for PURE, other extant RNR classes or reconstructed ancestral RNR variants, 61 which could be characterized with P-FLARE to assess their viability for metabolic expansion of PURE. Both ancestral and extant RNR classes may also provide viable starting points for the co-evolution of RNR and Phi29 DNAP and facilitate the synthesis of DNA (or u-DNA), without compromising on their respective catalytic activities. By adapting PURE encapsulation for evolution of self-encoded proteins, 25 RNR variants could therefore be co-evolved together with Phi29 DNAP to improve their interdependence and generate a minimal DNA metabolic pathway better matched to the capabilities of PURE. In its current form, the integration of an RNR Ia-based dNTP metabolic network into PURE has enabled the development of a system that reduces its heterotrophic reliance on external inputs to sustain the propagation of its genetic information. The direct expression of metabolic pathways in PURE may improve the self-sustainability and autonomy of recombinant expression systems, opening new avenues for cell-free systems that can dynamically respond to their metabolic demands and thereby extend their lifetime and robustness. 6 Future efforts to incorporate novel biosynthetic pathways, whether adapted from model organisms or evolved directly in PURE, may thus provide a new baseline for increasingly life-like autonomous synthetic systems. Methods DNA constructs All primers used for cloning and mutagenesis were ordered from IDT and are listed in Supplementary Tables 1 and 2, respectively. The coding sequences of E. coli nrdA (RNR Ia a), E. coli nrdB (RNR Ia b) and Bacillus phage phi29 Phi29 DNA polymerase can be found in Supplementary Table 3. General molecular biology techniques. PCRs for amplification and cloning of expression constructs were carried out using the Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs - NEB) using 500 nM of each primer. Sample preparation and thermocycling was carried out according to the manufacturer’s instructions. PCR products were confirmed with 1% TAE agarose gels and purified using the Monarch ® Spin PCR & DNA Cleanup Kit (5 μg) (NEB). DNA fragments were cloned into respective vectors using the NEBuilder HiFi DNA Assembly Master Mix (NEB) following the manufacturer's instructions and the resulting constructs were transformed in chemically competent Top10 cells prepared in house. Correct assemblies were confirmed by restriction mapping and by Sanger or Oxford Nanopore sequencing (Microsynth AG). Plasmids were isolated using the NucleoBond Xtra Midi kit (Machery-Nagel). Isolation of RNR Ia locus and RBS cloning. The genomic locus encoding both subunits of Escherichia coli ribonucleotide reductase 1a ( nrdA , Gene ID: 946612; nrdB , Gene ID: 946732) was isolated by colony PCR from E. coli Top10 cells (primers pr01 and pr02). The construct was cloned in cloned in a pBAD33 vector (backbone available in the lab and isolated with primer pr03 and pr04) to generate the pBAD33 RNR Ia MC (multi-cistron) construct and to obtain a consistent ribosome binding site (RBS) sequence. pET29b RNR expression vectors. Starting from the pBAD33 RNR MC plasmid, the pET29b RNR MC plasmid was constructed using primers pr05 and pr06 to isolate the cassette containing RBS and RNR locus and primers pr07 and pr08 for the pET29b backbone (available in the lab). Upstream of the RBS sequence, the constructs were optimized for in vitro translation by adding a T7 transcriptional promoter, followed by a T7 gene 10 translation-enhancer sequence 62 and a bidirectional transcription terminator downstream of the coding sequence. The resulting pET29b RNR MC was then used to create the individual pET29b RNR Alpha 6xHis and pET29b RNR Beta plasmids (with primer pairs pr09 – pr10 and pr11 – pr12, respectively). pUC19 RNR expression vectors. The expression cassettes (T7p, g10, RBS, nrdA or nrdB , TPhi terminator) were isolated from the individual pET29b RNR plasmids (in both cases with primers pr13 and pr14) and cloned into pUC19 (NEB) (backbone available in the lab and isolated with primers pr15 and pr16). Donated plasmids. The pET28b-nrdA and pTB-nrdB plasmids were kindly donated by JoAnne Stubbe and coworkers for purification of the individual RNR Ia subunits, as described in more detail in the Supplementary Methods. The pREP plasmid for Phi29 DNAP in vitro translation was generated as part of a previous study 21 and follows a similar cassette construction (i.e. T7 promoter, g10 leader sequence, RBS, Phi29 DNAP coding sequence and bi-directional TPhi terminator), optimized for in vitro transcription and translation. Single-stranded DNA oligos for P-FLARE assay. The DNA constructs for the P-FLARE assay were ordered from IDT and are listed in Supplementary Table 4. The ssDNA Broccoli construct was designed with a T7 promoter immediately upstream of the core sequence of the Broccoli fluorescent light-up aptamer, which lacked the F30 stem sequence, as described in more detail elsewhere. 38 Fluorescent Light-up Aptamer RNR Enzymatic assay in PURE (P-FLARE) P-FLARE assay reactions were carried out in the PURExpress (NEB) system in a final volume of 17 µL. A detailed list of components for a representative reaction can be found in Supplementary Table 5. A general reaction composition for both versions of the assay contained 1x (6.8 µL) PURExpress Solution A (NEB), 1x (5.1 µL) PURExpress Solution B (NEB); the volumes of PURExpress solutions were adjusted to the final reaction volume of the assay, while maintaining the same ratios specified in the manufacturer's instructions. P-FLARE reactions further contained 1 U/µL Murine Rnase Inhibitor (NEB), 10 µM DFHBI-1T (Biomol), 1.5 mM TCEP (Biotium), 10 U Phi29 DNA polymerase (NEB) and 500 nM of both Broccoli ssDNA and primer prBRO from an equimolar mix. To ensure in vitro translation of the RNR Ia heterodimer tetrameric complex, 4 nM of each pET29b RNR plasmid were supplied to the reactions, alongside 5 µM of ammonium iron(II) sulfate (prepared fresh each time) for correct folding of the b subunit and for formation of the Y 122 · radical in the b2 dimer. The same plasmid concentrations also applied when expressing only one RNR Ia subunits. When only a was expressed, 4 nM of pET29b RNR Alpha and 1 µM purified b2 was supplemented to the reaction. Before being added to the reactions, apo-b2 was pre-treated with 5 molar equivalents of ammonium iron(II) sulfate and incubated on ice for 10 minutes to ensure Y 122 · radical formation and generate the b2 to be added to the P-FLARE reactions. When only b was expressed in PURE, 4 nM of pET29b RNR Beta and 1 µM purified a2 was supplemented to the reaction. This general reaction framework was then used as the basis to test different combinations of deoxynucleoside triphosphates (dNTPs) (ThermoFisher Scientific). 200 µM of equimolar mixes with different combinations of dNTPs were supplied to the reactions to detect RNR-dependent reduction of the NDPs endogenously produced in PURE and not supplied as dNTPs. For example, when P-FLARE read-out depended on reduction of endogenous CDP, 200 µM of dDTPs were added (i.e. equimolar mixture of dATP, dGTP, dTTP). Instead, when supplying additional nucleoside diphosphates (NDPs) (CDP - TCI Chemicals, ADP - Jena Bioscience, GDP and UDP - SigmaAldrich) to P-FLARE in PURE, 2 mM of each freshly prepared NDP were supplied to the reactions in the specified combinations, on top of the nucleobases supplied as dNTPs. For example, if 2 mM CDP was supplied to the reaction, 200 µM of dDTPs were supplied alongside. Reactions were then gently mixed by pipetting and incubated at 30 ºC in a StepOne Real-Time PCR System (ThermoFisher Scientific) for 6 hours, with fluorescence measurements (EX 488 nm, EM 510 nm) every 6 minutes to reduce photobleaching of the DFHBI-1T fluorogen. Apparent rate estimation of P-FLARE assay The raw RFU data was normalized with min-max scaling between the averages of the internal negative and positive controls (i.e. ∆RNR, dDTPs - negative; ∆RNR, dNTPs - positive) and then smoothened with a Savitzky–Golay filter. The filtered data was then fit to a scaled logistic function. The apparent rate was then defined as the slope of a linear regression model fitted between the local maximum and minimum of the second derivative of the fitted logistic function. A more detailed explanation of the rate estimation is provided in the Supplementary Methods. Supplementary Fig. 20 provides an example image of the apparent rate estimation. RNR-dependent transcription-translation-coupled DNA replication Transcription-translation-coupled DNA replication (TTcDR) reactions were carried out in the TTcDR PURE system in a final volume of 12.5 µL. The composition of 10x D-EM is detailed in Supplementary Table 6 and was based on the TTcDR EM previously described 21,59 and was modified to accommodate RNR Ia's buffer requirements. A general reaction framework for TTcDR PURE was assembled as follows: 1x D-EM, 0.1x (0.5 µL) of PURExpress Solution A (NEB), 2x (7.5 µL) PURExpress Solution B (NEB), 1 U/µL Murine Rnase Inhibitor (NEB), 1.5 mM TCEP (Biotium) and 1x (0.25 µL) of 50x rNTP Mix (18.75 mM ATP, 12.5 mM GTP, 6.25 mM UTP and CTP – Supplementary Table 7). Furthermore, 3 nM of each pUC19 RNR plasmid were supplied to the reactions, alongside 5 µM of ammonium iron(II) sulfate and 600 µM of an equimolar mix of dNTPs in different combinations, depending on which endogenous NDPs had to be reduced. For example, if DNA replication depended on reduction of endogenous CDP, 600 µM of equimolar mixture of dDTPs were supplied to the reactions. A detailed list of components for a representative reaction can be found in Supplementary Table 8. Depending on the source of Phi29 DNAP, reactions were supplemented with either 10 U of purified Phi29 DNAP (NEB) or 3 nM of pREP plasmid. Reactions were gently mixed by pipetting and incubated for 16 hours at 30 ºC in a ProFlex thermocycler (ThermoFisher Scientific). In vivo propagation of RNR-dependent TTcDR products via C2C After incubation, TTcDR reactions were treated with 60 U (3 µL) of DpnI (NEB) and incubated at 37 ºC for 3 hours to digest input plasmids. To improve transformation efficiency, the plasmid concatemers generated by Phi29-dependent replication were first linearized with plasmid-specific single-cutter restriction enzymes, then re-circularized, with a circle-to-circle (C2C) protocol. To linearize the concatemers, after DpnI digestion, the TTcDR reactions were treated for 1 hour at 37 ºC with 20 U (1 µL) of both MluI-HF (NEB) and EcoRI-HF (NEB) (single cutter restriction enzymes for pUC19 RNR Alpha and pUC19 RNR Beta, respectively). While optimizing the C2C step, we consistently observed that heat-inactivating the restriction enzymes was necessary to improve ligation and transformation efficiency, as shown by exemplary data in Supplementary Fig. 21. The restriction enzymes were therefore heat inactivated by incubating the reactions at 85 ºC for 20 minutes. After concatemer linearization, 2 µL of Salt-T4 DNA Ligase (NEB) were added to the reactions, together with a final concentration of 1 mM ATP (Jena Bioscience) and incubated at 25 ºC for 16 hours. Final reaction volume prior to transformation is 20 µL. Final reactions were diluted 1:10 in ddH 2 O and kept at room temperature until transformation in 20 µL electrocompetent E. coli MegaX cells (DH10B T1R) (Invitrogen) according to the manufacturer’s protocol. 100 µL of recovery culture of the transformants were then grown overnight at 37 ºC on 1.5% LB-agar plates with 100 µg/mL carbenicillin resistance. When pREP was used as the source for Phi29 DNAP, 100 µL of recovery culture were also plated on 1.5% LB-agar plates with 37.5 µg/mL zeocine resistance. Plates were imaged in an Azure Biosystems Sapphire Imager at 488 nm excitation. Plate images were analyzed with ImageJ (Fiji v2.14.0) for colony counting after image thresholding. Each TTcDR reaction was repeated in three independent replicates, each of which was transformed three times to determine accuracy of transformation measurement. Confirmation of transformation of TTcDR products After transformation, sampled CFUs were picked for confirmation of the transformation of the representative pUC19 RNR Beta plasmid using Sanger sequencing (Microsynth AG). The remaining CFUs from the transformation of TTcDR products were re-suspended from the respective plates in 5 mL of LB medium and used to inoculate a 30 mL LB culture with the respective antibiotics (100 µg/mL carbenicillin for pUC19 RNR plasmids or 37.5 µg/mL zeocine for pREP) and grown overnight at 37 ºC. Plasmids were isolated from 5 mL at OD 600 = 4 of each culture using the NucleoSpin Plasmid, Mini kit (Machery-Nagel). 500 ng of isolated plasmids were then digested with 20 U (1 µL) of both MluI-HF (NEB) and EcoRI-HF (NEB) for 1 hour at 37 ºC. Restriction products were verified using a D5000 ScreenTape in a 4150 TapeStation System (Agilent). Booting of P-FLARE in PURE reactions with TTcDR products TTcDR reaction products were used as input for booting P-FLARE reactions in PURE. TTcDR reactions dependent on the in vitro translation of RNR Ia and reduction of endogenous NDPs were prepared as described above. Reactions were incubated at 30 ºC for 16 hours and then treated with 60 U of DpnI (NEB) for 3 hours at 37 ºC. P-FLARE reactions in PURE were prepared as described above, but instead of adding pET29b RNR plasmids, 2 µL of TTcDR reactions were added. P-FLARE reactions were then incubated for 6 hours at 30 ºC in a StepOne Real-Time PCR System (ThermoFisher Scientific) for 6 hours, with fluorescence measurements (EX 488 nm, EM 510 nm) every 6 minutes. Statistical analyses All sample sizes, error bars, and statistical tests are defined in figure legends. No statistical method was used to predetermine sample size. No data were excluded from the analyses. Analyses were performed with Python (v 3.13) and Excel (v 16.100.1). Statistical significance was defined as p < 0.05 (* - p ≤ 0.05; ** - p ≤ 0.01; *** - p ≤ 0.001). When multiple comparisons were performed within the same experimental set, p values were corrected with the Benjamini-Hochberg procedure (false discovery rate – FDR), with an alpha value of 0.05. When different P-FLARE reactions were compared with one another, the apparent reaction rates were compared with unpaired two-tailed Welch's t-tests. Comparisons were performed between apparent rates rather than maximum RFU values, since different reaction composition were observed to reach their maximum RFU values at different time points. Welch's t-tests were performed as equal variance could not be assumed either due to different sample composition or due to number of measurements to assume variance normality. When comparing transformation efficiency of different TTcDR reactions, the log-transformed CFU/mL counts of each replicate’s mean were compared to the ∆pUC-a negative control using a one-tailed two-sample unpaired Welch’s t-test to account for heteroscedasticity between samples. Declarations Author Information Corresponding authors Hannes Mutschler - Biomimetic Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, 44227, Germany; Email: [email protected] Author contributions H.M. and J.DC. conceived the project. J.DC. and N.E.N. designed experiments and collected data. J.DC. performed relevant analyses and designed the figures. V.G. purified RNR Ia subunits. All authors wrote the paper and approved the final manuscript for publication. Acknowledgements We would like to thank Alexander Wagner (TU Dortmund University, Germany) and Deni Szokoli (TU Dortmund University, Germany) for helpful discussions and Shari L. Meichsner (TU Dortmund University, Germany) for helpful support with the purification of the RNR Ia subunits. References Forster, A. C. & Church, G. M. Towards synthesis of a minimal cell. Mol Syst Biol 2 , 45 (2006). Jewett, M. C. & Forster, A. C. Update on designing and building minimal cells. Curr. Opin. 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A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J. Biol. Chem. 264 , 16973–16976 (1989). Additional Declarations There is NO Competing Interest. Supplementary Files DeCapitanietalSUPPLEMENTARYINFORMATION.pdf Supplementary Information GA.png Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9248024","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":619087049,"identity":"8f6bdca3-a13c-49f7-a76f-622cf7dae619","order_by":0,"name":"Hannes Mutschler","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8005-1657","institution":"TU Dortmund University","correspondingAuthor":true,"prefix":"","firstName":"Hannes","middleName":"","lastName":"Mutschler","suffix":""},{"id":619087050,"identity":"bc56d5d1-f621-47fa-9942-b87dd75b9d61","order_by":1,"name":"Jacopo De Capitani","email":"","orcid":"","institution":"TU Dortmund University","correspondingAuthor":false,"prefix":"","firstName":"Jacopo","middleName":"","lastName":"De Capitani","suffix":""},{"id":619087051,"identity":"a8c46fd9-acf0-4aa2-b208-82730ad05db3","order_by":2,"name":"Noemi Nwosu","email":"","orcid":"","institution":"TU Dortmund University","correspondingAuthor":false,"prefix":"","firstName":"Noemi","middleName":"","lastName":"Nwosu","suffix":""},{"id":619087052,"identity":"60b33048-e785-4b53-8468-70473cfe17be","order_by":3,"name":"Viktoria Gocke","email":"","orcid":"","institution":"TU Dortmund University","correspondingAuthor":false,"prefix":"","firstName":"Viktoria","middleName":"","lastName":"Gocke","suffix":""},{"id":619087053,"identity":"d06dbf04-68a2-4b53-b1a4-da013604c492","order_by":4,"name":"Muege Kasanmascheff","email":"","orcid":"","institution":"TU Dortmund University","correspondingAuthor":false,"prefix":"","firstName":"Muege","middleName":"","lastName":"Kasanmascheff","suffix":""}],"badges":[],"createdAt":"2026-03-27 20:05:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9248024/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9248024/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106403957,"identity":"35259b01-e587-4ff1-803c-a4f1cf085d8b","added_by":"auto","created_at":"2026-04-08 09:15:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":357492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegrating ribonucleotide reductase (RNR) Ia in PURE.\u003c/strong\u003e \u003cstrong\u003ea.\u003c/strong\u003e Schematic representation of the active RNR Ia a2b2 complex. The a2 dimer (blue) contains the allosteric regulatory sites (activity – green, and specificity – purple) and the catalytic site (yellow). The b2 dimer (orange) encompasses the di-ferric center (red) for the formation of the Y122· radical. Schematic based on PDB 6W4X.\u003csup\u003e39\u003c/sup\u003e \u003cstrong\u003eb.\u003c/strong\u003e Integrating RNR Ia in the PURE system. The genes encoding for RNR Ia are transcribed and translated by the extant PURE machinery. The resulting active RNR Ia a2b2 complex reduces the NDPs (nucleoside diphosphates) created from NTPs by the energy regeneration system native to PURE. The resulting dNDPs (deoxynucleoside diphosphates) are re-phosphorylated by nucleoside diphosphate kinase (NDK) into dNTPs (deoxynucleoside triphosphates). Phi29 DNA polymerase (DNAP) then uses the dNTPs to replicate the same starting plasmids encoding for RNR Ia.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9248024/v1/49a8e87a7e37b54e246758bf.png"},{"id":106414919,"identity":"d35240a2-4303-4d7b-ba7c-bc995c7abc83","added_by":"auto","created_at":"2026-04-08 10:30:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":350233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of RNR Ia activity in PURE with P-FLARE assay.\u003c/strong\u003e \u003cstrong\u003ea.\u003c/strong\u003e Read-out of RNR Ia activity in PURE using the Fluorescent Light-up Aptamer RNR Enzymatic assay (P-FLARE). The expressed RNR Ia a2b2 complex reduces NDPs to dNDPs, which are then re-phosphorylated by NDK. Phi29 DNAP incorporates the generated dNTPs (and any additional dNTPs supplied to the reaction) and extends the partially double stranded DNA template of the Broccoli fluorescent light-up aptamer (FLAP). The resulting dsDNA template is then transcribed by T7 RNA polymerase into the Broccoli FLAP, which, upon binding the DFHBI-1T fluorogen, can be detected by fluorescence emission. In this example, RNR Ia reduces CDP, and the reaction is supplemented with additional dDTPs (i.e. dATP, dGTP, dTTP). \u003cstrong\u003eb.\u003c/strong\u003e Expression of each RNR Ia subunit in PURE. Band volumes from Green\u003csub\u003eLys\u003c/sub\u003e-tRNA SDS-PAGE were normalized by number of Lys residues in each subunit and calculated as a function of the total of de novo\u003cem\u003e \u003c/em\u003eRNR Ia expression (see Supplementary Methods). \u003cstrong\u003ec.\u003c/strong\u003e Reaction matrix for panels d and e, for characterization of dCTP-limiting P-FLARE assay using RNR Ia partially or fully expressed in PURE. The colored boxes represent components included in the system. \u003cstrong\u003ed.\u003c/strong\u003e Normalized relative fluorescence units (RFU) read-out of P-FLARE reactions. P-FLARE reactions lacking RNR Ia either with all dNTPs (black) or with only dDTPs (gray) are compared with reactions where the signal depended on reduction of CDP upon expression of either a2 (blue), b2 (orange) or a2b2 (red). 2 mM CDP were supplemented to PURE for P-FLARE reactions \u003cstrong\u003ee.\u003c/strong\u003e Apparent rates of P-FLARE reactions shown in panel d. For panels d and e, data was normalized with min-max scaling between maximum value of the ∆RNR-dNTPs sample and t0 of the ∆RNR-dDTPs sample. For all panels, independent replicates were performed for all measurements, with n = 3. The mean and standard deviation are shown.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9248024/v1/5b72a7df0485d9facf0edd2f.png"},{"id":106404155,"identity":"5feb67dc-575c-435a-832a-382e25c8509b","added_by":"auto","created_at":"2026-04-08 09:15:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":231556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP-FLARE read-out for reduction of NDPs, either supplied or native to PURE. a.\u003c/strong\u003e Apparent rates of P-FLARE reactions. Reactions lacking RNR Ia expression with either all dNTPs (black) or only dDTPs (grey) are compared to reactions dependent on the reduction of an individual NDP by de novo\u003cem\u003e \u003c/em\u003eexpressed RNR Ia. Reactions with NDPs supplemented to PURE (red) (2 mM of each supplemented NDP) are compared to reactions where the same NDP is generated by the energy regeneration machinery of PURE (purple). \u003cstrong\u003eb.\u003c/strong\u003e Normalized real-time fluorescence read-out of P-FLARE reactions, dependent on the generation of dTTP (left) (either with 2 mM supplemented or endogenous UDP) or all dNTPs, synthesized by all NDPs generated in PURE (right). In the reaction matrices, colored boxes indicate components added to the reactions and shaded boxes indicate the respective NDPs supplemented to PURE. Data was normalized with min-max scaling between the maximum RFU values of the ∆RNR-dNTPs samples and t0 of ∆RNR-dDTPs sample. Welch’s t-test between a2b2-∆dNTPs sample and ∆RNR-dDTPs sample, p=0.006. Technical replicates were performed for all measurements, with n = 3. The mean and standard deviation are shown. Unpaired two-tailed Welch’s t-tests were used for pairwise comparisons between the different reaction rates and corrected for multiple comparisons with FDR.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9248024/v1/96d170cb51c88abb9941d47c.png"},{"id":106406019,"identity":"923f5169-347c-4966-9a2a-c28394a2777f","added_by":"auto","created_at":"2026-04-08 09:29:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":435441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNR-dependent replication of plasmids encoding RNR Ia during Transcription-Translation coupled DNA Replication (TTcDR) in PURE.\u003c/strong\u003e \u003cstrong\u003ea.\u003c/strong\u003eReaction matrix for the characterization of RNR-dependent DNA replication. Colored boxes indicate components added to the reactions. RNR-independent reactions supplied with all dNTPs (black) are compared to RNR-dependent reactions where DNA replication depends on reduction of endogenous CDP (blue), VDPs (i.e. ADP, GDP, CDP) (orange), or all NDPs (red). Negative controls: a2b2-∆Phi29-dNTPs (purple) and ∆pUCa-dDTPs (grey). \u003cstrong\u003eb.\u003c/strong\u003e Example plate images of transformed TTcDR products after circle-to-circle (C2C) re-circularization, shown as binary images after thresholding (Material and Methods). \u003cstrong\u003ec.\u003c/strong\u003e Colony-forming unit count per mL (CFU/mL) of transformed TTcDR products. Independent reaction replicates n=3. Each replicate is transformed 3 times. Overall mean of n=9 and SEM of individual replicate means shown, along with means of individual replicates. One-tailed unpaired two-sample Welch’s t-tests with ∆pUCa-dDTPs shown (a2b2-dDTPs vs ∆a-dDTPs, p=0.0004), (a2b2-dTTPs vs ∆a-dDTPs, p=0.002), (a2b2-∆dNTPs vs ∆a-dDTPs, p=0.0004). \u003cstrong\u003ed.\u003c/strong\u003e D5000 ScreenTape of restriction digestion product of pUC19 RNR plasmids isolated from re-suspended colonies. Expected sizes: pUC-a 4.9 kbp, pUC-b 3.7 kbp. Each reference pUC RNR plasmid is treated with both restriction enzymes. \u003cstrong\u003ee.\u003c/strong\u003eNormalized real-time fluorescence read-out of P-FLARE reactions using 2 µL of TTcDR reactions as DNA input for expression of RNR Ia. For panel e, independent replicates were performed, with n=3, mean and standard deviation shown.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9248024/v1/45357ba0483ea0c0ab1612f1.png"},{"id":106380587,"identity":"b97e5002-0610-44eb-aff0-104d872be395","added_by":"auto","created_at":"2026-04-08 05:02:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":653252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNA synthesis and replication dependent on expression of RNR Ia and Phi29 DNAP. a.\u003c/strong\u003eReaction matrix for the characterization of DNA synthesis and replication dependent on RNR Ia and Phi29 DNAP. Colored boxes indicate components added to the reactions. RNR-independent DNA synthesis with all dNTPs supplied (black) is compared to RNR-dependent reduction of endogenous CDP (blue), VDPs (ADP, GDP, CDP) (orange), or all NDPs (red). Negative controls: a2b2-∆Phi29-dNTPs (purple) and ∆pUCa-dDTPs (grey). \u003cstrong\u003eb.\u003c/strong\u003e Apparent rates of P-FLARE reactions dependent on Phi29 DNAP. Independent replicates were performed, with n=3. The mean and standard deviation are shown. Two-tailed unpaired Welch’s t-tests are shown, corrected for multiple comparisons with FDR (a2b2-Phi29-∆dNTPs vs a2b2-∆Phi29-dNTPs, p=0.022) (a2b2-Phi29-∆dNTPs vs ∆a-Phi29-dDTPs, p=0.022). \u003cstrong\u003ec.\u003c/strong\u003e Example plate images of TTcDR products based on Phi29 DNAP expression from pREP, and transformed after circle-to-circle (C2C) re-circularization, shown as binary images after thresholding. \u003cstrong\u003ed.\u003c/strong\u003e Colony-forming unit count per mL (CFU/mL) of transformed TTcDR products of pUC19 RNR Ia plasmids (plated on carbenicillin plates). One-tailed unpaired two-sample Welch’s t-tests with ∆pUCa-dDTPs shown (a2b2-dDTPs vs ∆a-dDTPs, p=0.0005), (a2b2-dTTPs vs ∆a-dDTPs, p=0.0003), (a2b2-∆dNTPs vs ∆a-dDTPs, p=0.001) \u003cstrong\u003ee.\u003c/strong\u003eCFU/mL of transformed TTcDR products of pREP plasmid (plated on zeocine plates). One-tailed unpaired two-sample Welch’s t-tests with ∆pUCa-dDTPs shown (a2b2-dDTPs vs ∆a-dDTPs, p=0.0009), (a2b2-dTTPs vs ∆a-dDTPs, p=0.0007), (a2b2-∆dNTPs vs ∆a-dDTPs, p=0.002). For panels d and e, independent replicates were performed, with n=3. Each replicate was transformed 3 times. Overall mean of n=9 and SEM of individual replicate means are shown, along with means of individual replicates. \u003cstrong\u003ef.\u003c/strong\u003e D5000 ScreenTape of restriction digestion product of pUC RNR plasmids isolated from re-suspended colonies. Expected sizes: pUC-a 4.9 kbp, pUC-b 3.7 kbp. \u003cstrong\u003eg.\u003c/strong\u003e D5000 ScreenTape of restriction digestion product of pREP plasmid isolated from re-suspended colonies. Expected pREP size 4.6 kbp. For panels f and g, each reference pUC RNR and pREP plasmid is treated with both restriction enzymes. \u003cstrong\u003eh.\u003c/strong\u003e Normalized real-time fluorescence read-out of P-FLARE reactions using 2 µL TTcDR reactions as DNA input for co-expression of RNR Ia and Phi29. For panel h, independent replicates were performed, with n=3. The mean and standard deviation are shown.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9248024/v1/1af6b59c8cfc81c78bce653f.png"},{"id":106724017,"identity":"27d9c2d0-cdaf-4dd7-ab25-0276a806c679","added_by":"auto","created_at":"2026-04-12 18:23:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2730455,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9248024/v1/39b7d3fc-27f5-43cd-aada-d65b04b09064.pdf"},{"id":106380585,"identity":"58af6492-ac62-456e-ba63-394c02892c72","added_by":"auto","created_at":"2026-04-08 05:02:18","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19918659,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"DeCapitanietalSUPPLEMENTARYINFORMATION.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9248024/v1/bc872de42955470996d5be03.pdf"},{"id":106404706,"identity":"8716e265-5628-4e3e-90f7-37adb62c3742","added_by":"auto","created_at":"2026-04-08 09:16:37","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8299477,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-9248024/v1/cd7fe21f971a56b8c1fa20e2.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Bootstrapping DNA replication with ribonucleotide reductase in a minimal cell-free system","fulltext":[{"header":"Introduction","content":"\u003cp\u003eArtificial minimal protein-based systems allow the study of complex dynamic biological processes in environments with controlled compositions.\u003csup\u003e1‑6\u003c/sup\u003e The PURE (Protein Synthesis Using Recombinant Elements) in vitro transcription-translation system has served as a minimal backbone to explore key reactions associated with the central dogma.\u003csup\u003e7,8\u003c/sup\u003e Notably, PURE can be augmented to display life-like processes outside of a cellular context, including self-regeneration of the translation machinery\u003csup\u003e9‑18\u003c/sup\u003e and replication\u003csup\u003e19‑22\u003c/sup\u003e and evolution\u003csup\u003e23‑27\u003c/sup\u003e of genetic information. Enabling these functions requires increased metabolic complexity within the standard PURE system.\u003csup\u003e28,29\u003c/sup\u003e Recent efforts have successfully integrated pathways for in situ synthesis of amino acids\u003csup\u003e30,31\u003c/sup\u003e and energy regeneration.\u003csup\u003e32‑34\u003c/sup\u003e However, the absence of metabolic complexity surrounding DNA replication represents a critical bottleneck that prevents the development of truly \u0026ldquo;life-like\u0026rdquo; minimal cell systems. Since PURE is an extremely heterotrophic multi-enzyme system, replication of its genetic content has remained dependent on the external supply of additional nucleobases or on the partial regeneration of nucleic acid building blocks through limited re-phosphorylation.\u003csup\u003e35‑37\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this work, we sought to augment PURE to enable the in situ\u003cem\u003e\u0026nbsp;\u003c/em\u003esynthesis of DNA building blocks and thereby reduce PURE\u0026rsquo;s dependency on external supply of DNA building blocks (Supplementary Fig. 1). We integrated the expression of \u003cem\u003eE. coli\u003c/em\u003e ribonucleotide reductase (RNR) Ia to synthesize dNTPs from available ribonucleotide pools, generated as part of the energy regeneration machinery in PURE. We verified the reliability of this system and confirmed that RNR Ia could efficiently supply all dNTPs for DNA synthesis, by coupling RNR Ia\u0026rsquo;s multi-turnover activity to the transcription of a fluorescent light-up RNA aptamer.\u003csup\u003e38\u003c/sup\u003e The synthesized dNTPs could be used to drive the replication of the plasmids encoding for a self-contained minimal DNA synthesis and replication metabolic network, encompassing the self-encoded RNR Ia and Phi29 DNA polymerase (DNAP).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCharacterization of RNR Ia activity in PURE using P-FLARE assay.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 261 kDa RNR Ia heterodimer tetrameric complex\u003csup\u003e39\u003c/sup\u003e can promiscuously reduce any nucleoside 5\u0026rsquo;-diphosphate (NDP) into its deoxynucleoside 5\u0026rsquo;-diphosphate (dNDP) form, through radical-based redox catalysis\u003csup\u003e40,41\u003c/sup\u003e and complex allosteric regulatory mechanisms of substrate and effector binding,\u003csup\u003e42,43\u003c/sup\u003e (Fig. 1a). Synthesis of the individual dNDPs by RNR Ia requires a sophisticated long-range, reversible Proton-Coupled Electron Transfer (PCET) as well as a complex orchestration of a series of intramolecular dynamics and efficient communication between the a2 and b2 homodimers. Upon coordination of Fe\u003csup\u003e2+\u003c/sup\u003e and formation of the Fe\u003csup\u003e3+\u003c/sup\u003e-Y\u003csub\u003e122\u003c/sub\u003e\u0026middot;\u0026nbsp;radical in the\u0026nbsp;b\u0026nbsp;subunits (Figure 1a),\u003csup\u003e41\u003c/sup\u003e the PCET between the b and a subunits\u003csup\u003e44,45\u003c/sup\u003e enables the generation the thyil radicals in the a subunit active site,\u003csup\u003e46\u003c/sup\u003e with which the assembled a2b2 complexes can reduce any NDP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe regulatory mechanism and multi-turnover reduction activity of RNR Ia in vitro depends on the presence of several cofactors, such as sufficient free Mg\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003eto facilitate intermolecular interactions between the\u0026nbsp;a2 and\u0026nbsp;b2 homodimers,\u003csup\u003e47\u003c/sup\u003e enough ATP to ensure the formation of the a2b2 active complex\u003csup\u003e48,49\u003c/sup\u003e and the optimal concentration of TCEP reducing agent to assist the regeneration of the catalytic cysteines in the a subunit.\u003csup\u003e50\u003c/sup\u003e The activity of RNR Ia could potentially be seamlessly integrated within the existing pathways of the PURE system (Fig. 1b), where NDPs are generated as part of aminoacylation, translation and regeneration of energy currencies. De novo synthesized RNR Ia could then use these NDPs to provide substrates to DNA synthesis by Phi29 DNA polymerase (DNAP). To probe if the reaction environment of the PURE system is compatible with these intricate enzymatic mechanisms and if it can sustain the multi-turnover reduction of different NDP substrates, we adapted the Fluorescent Light-up Aptamer RNR Enzymatic assay (FLARE)\u003csup\u003e51\u003c/sup\u003e for detection in PURE (i.e. P-FLARE) (Fig. 2a). The read-out of the FLARE assay relies on the reduction of NDPs by RNR Ia; the nucleoside diphosphate kinase (NDK) already present in PURE, phosphorylates the dNDPs generated by RNR Ia into dNTPs which can then be used by Phi29 DNAP to extend a partially double stranded DNA (dsDNA) template of the Broccoli fluorescent light-up aptamer (FLAP). Completion of this dsDNA template establishes a functional T7 promoter (T7p), enabling T7 RNA polymerase (RNAP) to transcribe the Broccoli FLAP, resulting in detectable green fluorescence upon binding of the fluorogen DFHBI-1T.\u003c/p\u003e\n\u003cp\u003eUsing P-FLARE, we confirmed that recombinant RNR Ia supplied to PURE could generate all sufficient dNDPs to generate a detectable signal (Supplementary Fig. 2). We then verified that expressed RNR Ia could assemble in its active\u0026nbsp;a2b2 complex from subunits expressed in similar rations from equimolar input plasmids (Fig. 2b and Supplementary Fig. 3). To test the RNR-dependent reduction of NDPs in PURE, we first adopted a dCTP-limiting set-up in P-FLARE (i.e. we supplied CDP and dDTPs \u0026ndash; dATP, dGTP, dTTP) to confirm that either one (Supplementary Fig. 4) or both de novo synthesized RNR Ia subunits (Fig. 2c) could generate the dCTP necessary for DNA synthesis. When either subunit was expressed alongside the other supplied purified subunit, the resulting\u0026nbsp;a2b2 complex reduces the CDP supplemented to PURE and helps generate the dCTP required for dsDNA synthesis and subsequent Broccoli FLAP transcription (Fig. 2d). However, when only the\u0026nbsp;a2 homodimer was expressed to form the complex with supplied purified\u0026nbsp;b2, this resulted in a lower maximum fluorescence signal (69%\u0026nbsp;\u0026plusmn;\u0026nbsp;6% of RNR-independent P-FLARE), likely due to suboptimal subunit ratio between expressed\u0026nbsp;a2 and supplied\u0026nbsp;b2.\u003csup\u003e48\u003c/sup\u003e When both subunits are expressed simultaneously in roughly equimolar ratios, the fluorescent signal reached similar intensities as in RNR-independent reactions, while incurring in a signal delay due to the necessary expression of both subunits (Fig. 2d). The observed P-FLARE rates are comparable irrespective of which subunit is expressed, which suggested that the formation of the active RNR Ia complex was the rate limiting step for the assay\u0026rsquo;s read-out (Fig. 2e). Using this dCTP-limiting set-up, we crucially confirmed that the observed fluorescent signals depended on de novoRNR Ia correctly generating the Fe\u003csup\u003e3+\u003c/sup\u003e-Y\u003csub\u003e122\u003c/sub\u003e\u0026middot; radical (Supplementary Fig. 5) and establishing a functional PCET between the two homodimers (Supplementary Fig. 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishing deoxynucleotide metabolism in PURE using RNR Ia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then investigated the synthesis of dNTPs from NDPs endogenously generated in PURE. The dephosphorylation of NTPs by NDK during energy regeneration produces enough NDPs to serve as a substrate for RNR Ia for the reduction into dNDPs. These are subsequently re-phosphorylated by NDK into dNTPs that can be utilized by Phi29 DNAP to synthesize new DNA. By testing different input concentrations of CDP in a dCTP-limiting P-FLARE assay, we observed that P-FLARE rates were consistently independent of exogenously supplied CDP (Supplementary Fig. 7). Using P-FLARE reactions with specific dNTP-limiting conditions (e.g. when ADP had to be reduced, we added only dBTPs \u0026ndash; dGTP, dCTP, dTTP), we confirmed that each of the four NDPs endogenously generated in PURE could be reduced by RNR Ia at similar rates to when the same NDP was supplemented to the reactions. (Fig. 3a and Supplementary Fig. 8). Encouragingly, P-FLARE reactions displayed similar rates when read-out depended on the reduction of either ADP or GDP, with a slight decrease when it depended on CDP reduction. In contrast, reactions that required the synthesis dUTP and, subsequently, uracil-DNA (u-DNA) for read-out, showed starkly diminished read-out signals (Fig. 3b and Supplementary Fig. 8c). This decrease in RNA synthesis could be a result of lower incorporation rates of dUTP by Phi29 DNAP or reduced dUTP synthesis rates by RNR Ia. This effect was to be expected given that in living cells dUTP is usually converted into dTTP by thymidylate synthase and thymidylate kinase.\u003csup\u003e52‑54\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eTo disentangle these potentially compounding effects, we tested P-FLARE read-out hinging on the synthesis of u-DNA in the presence of supplemented dUTP (Supplementary Fig. 9). In this set-up we observed lower rates and Broccoli yields when dUTP was supplemented instead of dTTP in RNR-independent P-FLARE read-outs, which can be explained by lower incorporation rates of dUTP by Phi29 DNAP, as previously observed.\u003csup\u003e51\u003c/sup\u003e Instead, when P-FLARE read-out depended on the activity of both Phi29 DNAP and RNR Ia, lower concentrations of dUTP synthesized by RNR Ia resulted in a further decrease in RNA synthesis. As such, the coupled activity of RNR Ia and Phi29 DNAP guaranteed that when no dNTPs or NDPs were supplied to PURE, RNR Ia could supply all necessary dNTPs for DNA synthesis, despite a compounding metabolic effect (Fig. 3b, Supplementary Fig. 8c and Supplementary Fig. 9).\u003c/p\u003e\n\u003cp\u003eThe fact that newly synthesizedRNR Ia could convert all endogenous NDPs into their respective dNDPs was a crucial step for the development of a self-contained DNA synthesis metabolic network. This observation suggested that de novoRNR Ia maintained the allosteric regulation in which specific reduced dNTPs function as the effectors for the reduction of subsequent NDPs, following a \u0026ldquo;reduction cascade\u0026rdquo;.\u003csup\u003e43\u003c/sup\u003e Using P-FLARE in PURE, we mimicked the intracellular regulation of de novo DNA building by simulating this precise regulatory pattern observed in mechanistic studies\u003csup\u003e42,43\u003c/sup\u003e (Supplementary Fig. 10). We probed RNR Ia reduction of various endogenous NDPs using all 16 possible input dNTP combinations (Supplementary Fig. 11). Reaction rates were generally consistent irrespective of which NDP combination had to be reduced, apart from when dUTP had to be synthesized from available UDP, which resulted in lower downstream transcription of Broccoli FLAP. As we observed previously,\u003csup\u003e51\u003c/sup\u003e RNR Ia relies on available dTTP as the specificity effector for dGTP generation. Nonetheless, in the absence of dTTP, RNR Ia notably appeared to be capable of using dUTP as a weak-specificity effector to initiate GDP reduction instead of dTTP. This is evidenced by the detection of reduced but highly significant (p=0.003) P-FLARE activity in all samples where both dGTP and dTTP were omitted (see Supplementary Fig. 11). Therefore, similarly as in deoxynucleotide de novobiosynthetic pathways in \u003cem\u003eE.\u0026nbsp;\u003c/em\u003ecoli,\u003csup\u003e55‑57\u003c/sup\u003e in PURE, RNA building blocks (i.e. NTPs) are de-phosphorylated by NDK into NDPs, which can serve as substrate for RNR Ia for in situsynthesis of all dNTPs, which can subsequently be incorporated into nascent DNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAutocatalytic RNR-dependent DNA replication of plasmids encoding RNR Ia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter confirming that all dNTPs could be synthesized in situin PURE by de novo expressed RNR Ia, we coupled RNR Ia activity to the replication of its encoding pUC-a\u0026nbsp;and pUC-b\u0026nbsp;plasmids (Supplementary Fig. 12). We designed this metabolic pathway so that Phi29 DNAP could rely solely on dNTPs generated by self-encodedRNR Ia starting only from endogenous NDPs. With this system, we therefore coupled the metabolic activity of RNR Ia to the autocatalytic propagation of its own genetic information. Rolling-circle amplification (RCA) of the pUC RNR vectors relied on RNA primers being generated by the exonuclease activity of Phi29 DNAP, therefore reducing the heterotrophic dependency on external input of additional Phi29-related proteins or exonuclease-resistant DNA primers.\u003csup\u003e19,20,58\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eWe first optimized a composition of PURE to couple the expression of RNR Ia with whole-plasmid replication (i.e. transcription-translation-coupled DNA replication \u0026ndash; TTcDR PURE).\u003csup\u003e19,21,59\u003c/sup\u003e After adjusting the formulation of TTcDR PURE to accommodate the required reducing agent for RNR Ia\u003csup\u003e50\u003c/sup\u003e (Supplementary Fig. 13), we observed that TTcDR PURE was less sensitive than PURExpress when used for the P-FLARE assay (Supplementary Fig. 14), but it substantially improved replication of pUC RNR vectors compared to PURExpress (Supplementary Fig. 15).\u003c/p\u003e\n\u003cp\u003eUsing this optimized TTcDR PURE composition, we confirmed strictly RNR-dependent TTcDR of the input plasmids by transforming the DNA resulting from TTcDR in \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003ecells (Fig. 4a, 4b). Replication of both pUC-a\u0026nbsp;and pUC-b\u0026nbsp;plasmids depended on the synthesis of either dCTP (i.e. supplying dDTPs), dVTPs (i.e. supplying only dTTP), or all dNTPs by de novoRNR Ia. The transformation of the products of DNA replication resulted in ~10\u003csup\u003e4\u003c/sup\u003e colony-forming units per mL (CFUs/mL), when both pUC-a and pUC-b plasmids were supplied. In contrast, the number of background CFUs/mL was reduced to less than 10\u003csup\u003e2\u003c/sup\u003e when either pUC-a or Phi29 DNAP were omitted from the reactions. (Fig. 4b, 4c). As such, this semi-quantitative read-out of plasmid replication, demonstrated that the efficiency of RNR-dependent TTcDR did not hinge on the number of unique dNTPs that had to be synthesized by RNR Ia. We further confirmed that the transformed and propagated TTcDR products contained full-length plasmid sequences using restriction mapping and Sanger sequencing of isolated of pUC-b plasmid DNA from sampled colonies (Fig. 4d and Supplemental Data).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further validate the replication of the genetic information encoding for RNR Ia, we adapted the P-FLARE assay to rely on the expression of functional RNR Ia starting from the DNA generated during TTcDR (Fig. 4e). Specifically, the expression of RNR Ia, and hence the P-FLARE read-out, depended on the amounts of RNR Ia-encoding DNA produced by the upstream TTcDR reaction, which on the other hand was dependent on the reduction of either dCTP, dVTPs or all dNTPs. After extensive DpnI incubation to remove the parental pUC-a and pUC-b plasmids, a fraction of the TTcDR reaction was used as input for P-FLARE reactions in PURE (Fig. 4e). We anticipated that increased DNA replication yields in TTcDR would result in higher expression yields of RNR Ia in P-FLARE, which in turn would result in increased amounts of dsDNA template generated by Phi29 DNAP and therefore enhanced transcription yields of Broccoli FLAP by T7 RNAP. We recorded a significant increase of Broccoli fluorescence in P-FLARE reactions supplied with the DNA products from TTcDR reactions dependent on dCTP synthesis by the self-encoded RNR Ia. Here, fluorescence readouts reached 35 % \u0026plusmn; 3% of the P-FLARE reactions supplied with the products of the RNR-independent TTcDR (i.e. supplied with all four dNTPs) (Fig. 4e). In contrast, when the upstream TTcDR reactions relied on the synthesis of dVTPs or all dNTPs, we detected only background-level signals. Presumably, the amount of DNA produced in these TTcDR reactions, when diluted in a new P-FLARE reaction, was not sufficient to generate enough functional RNR Ia to kickstart Broccoli transcription in P-FLARE within the lifespan of a bulk transcription-translation reaction in PURE. However, this crucially confirmed that while RNR Ia was expressed at sufficient levels to generate the necessary dNTPs for plasmid replication in TTcDR, the carry-over of expressed RNR Ia and synthesized dNTPs were insufficient to elicit a signal in the downstream P-FLARE reaction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-expression of RNR Ia and Phi29 DNAP enables autocatalytic synthesis and replication of minimal DNA replication machinery.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, we assessed whether RNR-dependent DNA replication could be introduced in PURE to recreate a more life-like replication of DNA, by mimicking intracellular DNA synthesis where dNTPs are generated by de novobiosynthetic pathways and used for replication of the genetic information encoding for those same pathways. We approached this challenge by designing a system where the activity of a minimal DNA replication machinery, comprising both self-encoded RNR Ia and Phi29 DNAP, drive the replication and propagation of the 13 kbp minimal genome encompassing the metabolic pathway.\u003c/p\u003e\n\u003cp\u003eUsing P-FLARE, we first confirmed that when co-expressing both proteins, de novoPhi29 DNAP could efficiently incorporate all dNTPs generated by RNR Ia (Fig. 5a, 5b), and that observed rates where comparable if Phi29 DNAP was either supplied or expressed from the pREP plasmid (Supplementary Fig. 16). Encouragingly, P-FLARE reactions generated detectable signals even when RNR Ia was required to synthesize an increasing number of dNTPs, although reaction rates gradually declined, likely due to reduced translation yields of both proteins and an increased metabolic burden on RNR Ia (Fig. 5b and Supplementary Fig. 17).\u003c/p\u003e\n\u003cp\u003eUsing the same reaction set-ups lacking specific or all dNTPs (Fig. 5a), we confirmed that this minimal self-encoded DNA building pathway expressed in PURE could generate all dNTPs in situand utilize them to replicate all the plasmids encoding for this pathway (Fig. 5c and Supplementary Fig. 18). The replication products of all three input plasmids encoding the minimal autocatalytic TTcDR system (pUC-a, pUC-b\u0026nbsp;and pREP) retained their ability for in vivo propagation since transformation of the products of DNA replication resulted in ~ 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eto 10\u003csup\u003e5\u003c/sup\u003e CFUs/mL when using selection markers for either the pUC RNR plasmids or pREP (Fig. 5d, 5e). As anticipated, the efficiency of TTcDR was not limited by how many dNTPs had to be synthesized or by the source of Phi29 DNAP, but rather by the activity of expressed RNR Ia (Supplementary Fig. 19). We also confirmed that the transformed and propagated TTcDR products contained full-length plasmid sequences of all three plasmids using restriction mapping and Sanger sequencing of isolated of pUC-b plasmid DNA from sampled colonies (Fig. 5f, 5g and Supplemental Data). In contrast, no background colonies were observed when either pUC-a or pREP were omitted from the reactions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEncouragingly, by kickstarting P-FLARE reactions with DNA replicated with our minimal self-contained DNA synthesis pathway, we confirmed that the concentrations of plasmids replicated during TTcDR followed a similar pattern when Phi29 DNAP was either supplied or co-expressed (Fig. 4a and 5h respectively). When the pUC RNR Ia and pREP vectors, replicated by de novoexpressed Phi29 DNAP, were used to reboot the synthesis of both proteins in P-FLARE, the generated amounts of replicated plasmids could sustain the synthesis of DNA in a dCTP-limiting set-up, reaching 34% \u0026plusmn; 5% of the RNR-independent reactions (Fig. 5h). Therefore, the in situsynthesis of dNTPs by RNR Ia created a rate-limiting metabolic step for DNA replication, akin to what is observed in vivoin \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003ecells as part of metabolic pathwaysfor de novodNTP biosynthesis.\u003csup\u003e55‑57\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we set out to expand the metabolic capabilities of PURE to advance the development of increasingly autonomous artificial systems able to produce components required for their own self-regeneration and propagation.\u003csup\u003e60\u003c/sup\u003e Starting only from the ribonucleotide pools available in PURE, we reconstructed a de novo dNTP\u003cem\u003e\u0026nbsp;\u003c/em\u003ebiosynthetic metabolic pathway driven by the expression of RNR Ia. We then integrated RNR Ia activity into a self-contained, autocatalytic DNA synthesis pathway together with Phi29 DNAP, enabling faithful replication of its own genetic and re-transformation and propagation in \u003cem\u003eE. coli\u003c/em\u003e cells. The integration of RNR Ia into PURE therefore represented an important step towards the development of a metabolically self-sufficient TTcDR system. Such a system would rely on its own components to replicate its own DNA while using the allosteric regulation of RNR Ia to dynamically tune dNTP production for continued propagation.\u003csup\u003e5,28\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eSeveral challenges remain to improve the self-sufficiency and self-regenerative capabilities of the system. Competition of limited resources between transcription, translation and DNA replication likely reduces RNR Ia yields,\u003csup\u003e59\u003c/sup\u003e limits the availability of NDP substrates, and decreased DNA replication rates. As a result, compared with reactions where all dNTPs were supplied exogenously, RNR-dependent reactions showed a 30-40% decrease in reaction rates in P-FLARE reactions and about 35% fewer colonies observed during plasmid in vivo\u003cem\u003e\u0026nbsp;\u003c/em\u003epropagation. These effects are likely the result of mismatch between the expressed RNR Ia and the regeneration of its required cofactors (i.e. ATP regeneration and free Mg\u003csup\u003e2+\u003c/sup\u003e) in PURE, or sub-optimal stoichiometries between the expressed RNR Ia subunits, both of which could in turns lead to unfavorable ratios of active and inactive RNR Ia complexes.\u003csup\u003e48,49\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAddressing these constraints will likely require optimization of the expression levels of the RNR Ia subunits and of Phi29 DNAP to improve dNTP and DNA synthesis. In addition, incorporating a dTTP synthesis pathway into PURE will help to overcome the current limitation of u-DNA synthesis. Including thymidylate synthase in PURE will further improve the allosteric regulation RNR Ia and the efficiency of nucleotide incorporation rate by Phi29 DNAP relative to dUTP. Alternatively, the integration of dNTP metabolism in PURE may benefit from the use of RNR Ia variants optimized for PURE, other extant RNR classes or reconstructed ancestral RNR variants,\u003csup\u003e61\u003c/sup\u003e which could be characterized with P-FLARE to assess their viability for metabolic expansion of PURE. Both ancestral and extant RNR classes may also provide viable starting points for the co-evolution of RNR and Phi29 DNAP and facilitate the synthesis of DNA (or u-DNA), without compromising on their respective catalytic activities. By adapting PURE encapsulation for evolution of self-encoded proteins,\u003csup\u003e25\u003c/sup\u003e RNR variants could therefore be co-evolved together with Phi29 DNAP to improve their interdependence and generate a minimal DNA metabolic pathway better matched to the capabilities of PURE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn its current form, the integration of an RNR Ia-based dNTP metabolic network into PURE has enabled the development of a system that reduces its heterotrophic reliance on external inputs to sustain the propagation of its genetic information. The direct expression of metabolic pathways in PURE may improve the self-sustainability and autonomy of recombinant expression systems, opening new avenues for cell-free systems that can dynamically respond to their metabolic demands and thereby extend their lifetime and robustness.\u003csup\u003e6\u003c/sup\u003e Future efforts to incorporate novel biosynthetic pathways, whether adapted from model organisms or evolved directly in PURE, may thus provide a new baseline for increasingly life-like autonomous synthetic systems.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eDNA constructs\u003c/h2\u003e\n\u003cp\u003eAll primers used for cloning and mutagenesis were ordered from IDT and are listed in Supplementary Tables 1 and 2, respectively. The coding sequences of \u003cem\u003eE. coli\u003c/em\u003e \u003cem\u003enrdA\u003c/em\u003e (RNR Ia\u0026nbsp;a),\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e \u003cem\u003enrdB\u003c/em\u003e (RNR Ia\u0026nbsp;b) and \u003cem\u003eBacillus phage phi29\u003c/em\u003e Phi29 DNA polymerase can be found in Supplementary Table 3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral molecular biology techniques.\u0026nbsp;\u003c/strong\u003ePCRs for amplification and cloning of expression constructs were carried out using the Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs - NEB) using 500 nM of each primer. Sample preparation and thermocycling was carried out according to the manufacturer\u0026rsquo;s instructions. PCR products were confirmed with 1% TAE agarose gels and purified using the Monarch\u003csup\u003e\u0026reg;\u003c/sup\u003e Spin PCR \u0026amp; DNA Cleanup Kit (5 \u0026mu;g) (NEB).\u003c/p\u003e\n\u003cp\u003eDNA fragments were cloned into respective vectors using the NEBuilder HiFi DNA Assembly Master Mix (NEB) following the manufacturer\u0026apos;s instructions and the resulting constructs were transformed in chemically competent Top10 cells prepared in house.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCorrect assemblies were confirmed by restriction mapping and by Sanger or Oxford Nanopore sequencing (Microsynth AG). Plasmids were isolated using the NucleoBond Xtra Midi kit (Machery-Nagel).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of RNR Ia locus and RBS cloning.\u003c/strong\u003e The genomic locus encoding both subunits of \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eribonucleotide reductase 1a (\u003cem\u003enrdA\u003c/em\u003e, Gene ID: 946612; \u003cem\u003enrdB\u003c/em\u003e, Gene ID: 946732) was isolated by colony PCR from \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eTop10 cells (primers pr01 and pr02). The construct was cloned in cloned in a pBAD33 vector (backbone available in the lab and isolated with primer pr03 and pr04) to generate the pBAD33 RNR Ia MC (multi-cistron) construct and to obtain a consistent ribosome binding site (RBS) sequence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003epET29b RNR expression vectors.\u003c/strong\u003e Starting from the pBAD33 RNR MC plasmid, the pET29b RNR MC plasmid was constructed using primers pr05 and pr06 to isolate the cassette containing RBS and RNR locus and primers pr07 and pr08 for the pET29b backbone (available in the lab). Upstream of the RBS sequence, the constructs were optimized for in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003etranslation by adding a T7 transcriptional promoter, followed by a T7 gene 10 translation-enhancer sequence \u003csup\u003e62\u003c/sup\u003e and a bidirectional transcription terminator downstream of the coding sequence. The resulting pET29b RNR MC was then used to create the individual pET29b RNR Alpha 6xHis and pET29b RNR Beta plasmids (with primer pairs pr09 \u0026ndash; pr10 and pr11 \u0026ndash; pr12, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003epUC19 RNR expression vectors.\u0026nbsp;\u003c/strong\u003eThe expression cassettes (T7p, g10, RBS, \u003cem\u003enrdA\u003c/em\u003e or \u003cem\u003enrdB\u003c/em\u003e, TPhi terminator) were isolated from the individual pET29b RNR plasmids (in both cases with primers pr13 and pr14) and cloned into pUC19 (NEB) (backbone available in the lab and isolated with primers pr15 and pr16).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDonated plasmids.\u003c/strong\u003e The pET28b-nrdA and pTB-nrdB plasmids were kindly donated by JoAnne Stubbe and coworkers for purification of the individual RNR Ia subunits, as described in more detail in the Supplementary Methods. The pREP plasmid for Phi29 DNAP in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003etranslation was generated as part of a previous study\u003csup\u003e21\u003c/sup\u003e and follows a similar cassette construction (i.e. T7 promoter, g10 leader sequence, RBS, Phi29 DNAP coding sequence and bi-directional TPhi terminator), optimized for in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003etranscription and translation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-stranded DNA oligos for P-FLARE assay.\u003c/strong\u003e The DNA constructs for the P-FLARE assay were ordered from IDT and are listed in Supplementary Table 4. The ssDNA Broccoli construct was designed with a T7 promoter immediately upstream of the core sequence of the Broccoli fluorescent light-up aptamer, which lacked the F30 stem sequence, as described in more detail elsewhere.\u003csup\u003e38\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003eFluorescent Light-up Aptamer RNR Enzymatic assay in PURE (P-FLARE)\u003c/h2\u003e\n\u003cp\u003eP-FLARE assay reactions were carried out in the PURExpress (NEB) system in a final volume of 17 \u0026micro;L. A detailed list of components for a representative reaction can be found in Supplementary Table 5. A general reaction composition for both versions of the assay contained 1x (6.8 \u0026micro;L) PURExpress Solution A (NEB), 1x (5.1 \u0026micro;L) PURExpress Solution B (NEB); the volumes of PURExpress solutions were adjusted to the final reaction volume of the assay, while maintaining the same ratios specified in the manufacturer\u0026apos;s instructions. P-FLARE reactions further contained 1 U/\u0026micro;L Murine Rnase Inhibitor (NEB), 10 \u0026micro;M DFHBI-1T (Biomol), 1.5 mM TCEP (Biotium), 10 U Phi29 DNA polymerase (NEB) and 500 nM of both Broccoli ssDNA and primer prBRO from an equimolar mix.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo ensure in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003etranslation of the RNR Ia heterodimer tetrameric complex, 4 nM of each pET29b RNR plasmid were supplied to the reactions, alongside 5 \u0026micro;M of ammonium iron(II) sulfate (prepared fresh each time) for correct folding of the\u0026nbsp;b\u0026nbsp;subunit and for formation of the Y\u003csub\u003e122\u003c/sub\u003e\u0026middot;\u0026nbsp;radical in the\u0026nbsp;b2 dimer. The same plasmid concentrations also applied when expressing only one RNR Ia subunits. When only\u0026nbsp;a\u0026nbsp;was expressed, 4 nM of pET29b RNR Alpha and 1 \u0026micro;M purified\u0026nbsp;b2 was supplemented to the reaction. Before being added to the reactions, apo-b2 was pre-treated with 5 molar equivalents of ammonium iron(II) sulfate and incubated on ice for 10 minutes to ensure Y\u003csub\u003e122\u003c/sub\u003e\u0026middot;\u0026nbsp;radical formation and generate the\u0026nbsp;b2 to be added to the P-FLARE reactions. When only\u0026nbsp;b\u0026nbsp;was expressed in PURE, 4 nM of pET29b RNR Beta and 1 \u0026micro;M purified\u0026nbsp;a2 was supplemented to the reaction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis general reaction framework was then used as the basis to test different combinations of deoxynucleoside triphosphates (dNTPs) (ThermoFisher Scientific). 200 \u0026micro;M of equimolar mixes with different combinations of dNTPs were supplied to the reactions to detect RNR-dependent reduction of the NDPs endogenously produced in PURE and not supplied as dNTPs. For example, when P-FLARE read-out depended on reduction of endogenous CDP, 200 \u0026micro;M of dDTPs were added (i.e. equimolar mixture of dATP, dGTP, dTTP). Instead, when supplying additional nucleoside diphosphates (NDPs) (CDP - TCI Chemicals, ADP - Jena Bioscience, GDP and UDP - SigmaAldrich) to P-FLARE in PURE, 2 mM of each freshly prepared NDP were supplied to the reactions in the specified combinations, on top of the nucleobases supplied as dNTPs. For example, if 2 mM CDP was supplied to the reaction, 200 \u0026micro;M of dDTPs were supplied alongside.\u003c/p\u003e\n\u003cp\u003eReactions were then gently mixed by pipetting and incubated at 30 \u0026ordm;C in a StepOne Real-Time PCR System (ThermoFisher Scientific) for 6 hours, with fluorescence measurements (EX 488 nm, EM 510 nm) every 6 minutes to reduce photobleaching of the DFHBI-1T fluorogen.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eApparent rate estimation of P-FLARE assay\u003c/h2\u003e\n\u003cp\u003eThe raw RFU data was normalized with min-max scaling between the averages of the internal negative and positive controls (i.e. ∆RNR, dDTPs - negative; ∆RNR, dNTPs - positive) and then smoothened with a Savitzky\u0026ndash;Golay filter. The filtered data was then fit to a scaled logistic function. The apparent rate was then defined as the slope of a linear regression model fitted between the local maximum and minimum of the second derivative of the fitted logistic function. A more detailed explanation of the rate estimation is provided in the Supplementary Methods. Supplementary Fig. 20 provides an example image of the apparent rate estimation.\u003c/p\u003e\n\u003ch2\u003eRNR-dependent transcription-translation-coupled DNA replication\u003c/h2\u003e\n\u003cp\u003eTranscription-translation-coupled DNA replication (TTcDR) reactions were carried out in the TTcDR PURE system in a final volume of 12.5 \u0026micro;L. The composition of 10x D-EM is detailed in Supplementary Table 6 and was based on the TTcDR EM previously described\u003csup\u003e21,59\u003c/sup\u003e and was modified to accommodate RNR Ia\u0026apos;s buffer requirements. A general reaction framework for TTcDR PURE was assembled as follows: 1x D-EM, 0.1x (0.5 \u0026micro;L) of PURExpress Solution A (NEB), 2x (7.5 \u0026micro;L) PURExpress Solution B (NEB), 1 U/\u0026micro;L Murine Rnase Inhibitor (NEB), 1.5 mM TCEP (Biotium) and 1x (0.25 \u0026micro;L) of 50x rNTP Mix (18.75 mM ATP, 12.5 mM GTP, 6.25 mM UTP and CTP \u0026ndash; Supplementary Table 7). Furthermore, 3 nM of each pUC19 RNR plasmid were supplied to the reactions, alongside 5 \u0026micro;M of ammonium iron(II) sulfate and 600 \u0026micro;M of an equimolar mix of dNTPs in different combinations, depending on which endogenous NDPs had to be reduced. For example, if DNA replication depended on reduction of endogenous CDP, 600 \u0026micro;M of equimolar mixture of dDTPs were supplied to the reactions. A detailed list of components for a representative reaction can be found in Supplementary Table 8.\u003c/p\u003e\n\u003cp\u003eDepending on the source of Phi29 DNAP, reactions were supplemented with either 10 U of purified Phi29 DNAP (NEB) or 3 nM of pREP plasmid. Reactions were gently mixed by pipetting and incubated for 16 hours at 30 \u0026ordm;C in a ProFlex thermocycler (ThermoFisher Scientific).\u003c/p\u003e\n\u003ch2\u003eIn vivo propagation of RNR-dependent TTcDR products via C2C\u003c/h2\u003e\n\u003cp\u003eAfter incubation, TTcDR reactions were treated with 60 U (3 \u0026micro;L) of DpnI (NEB) and incubated at 37 \u0026ordm;C for 3 hours to digest input plasmids. To improve transformation efficiency, the plasmid concatemers generated by Phi29-dependent replication were first linearized with plasmid-specific single-cutter restriction enzymes, then re-circularized, with a circle-to-circle (C2C) protocol. To linearize the concatemers, after DpnI digestion, the TTcDR reactions were treated for 1 hour at 37 \u0026ordm;C with 20 U (1 \u0026micro;L) of both MluI-HF (NEB) and EcoRI-HF (NEB) (single cutter restriction enzymes for pUC19 RNR Alpha and pUC19 RNR Beta, respectively). While optimizing the C2C step, we consistently observed that heat-inactivating the restriction enzymes was necessary to improve ligation and transformation efficiency, as shown by exemplary data in Supplementary Fig. 21. The restriction enzymes were therefore heat inactivated by incubating the reactions at 85 \u0026ordm;C for 20 minutes. After concatemer linearization, 2 \u0026micro;L of Salt-T4 DNA Ligase (NEB) were added to the reactions, together with a final concentration of 1 mM ATP (Jena Bioscience) and incubated at 25 \u0026ordm;C for 16 hours. Final reaction volume prior to transformation is 20 \u0026micro;L.\u003c/p\u003e\n\u003cp\u003eFinal reactions were diluted 1:10 in ddH\u003csub\u003e2\u003c/sub\u003eO and kept at room temperature until transformation in 20 \u0026micro;L electrocompetent \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eMegaX cells (DH10B T1R) (Invitrogen) according to the manufacturer\u0026rsquo;s protocol. 100 \u0026micro;L of recovery culture of the transformants were then grown overnight at 37 \u0026ordm;C on 1.5% LB-agar plates with 100 \u0026micro;g/mL carbenicillin resistance. When pREP was used as the source for Phi29 DNAP, 100 \u0026micro;L of recovery culture were also plated on 1.5% LB-agar plates with 37.5 \u0026micro;g/mL zeocine resistance. Plates were imaged in an Azure Biosystems Sapphire Imager at 488 nm excitation. Plate images were analyzed with ImageJ (Fiji v2.14.0) for colony counting after image thresholding. Each TTcDR reaction was repeated in three independent replicates, each of which was transformed three times to determine accuracy of transformation measurement.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConfirmation of transformation of TTcDR products\u003c/h2\u003e\n\u003cp\u003eAfter transformation, sampled CFUs were picked for confirmation of the transformation of the representative pUC19 RNR Beta plasmid using Sanger sequencing (Microsynth AG). The remaining CFUs from the transformation of TTcDR products were re-suspended from the respective plates in 5 mL of LB medium and used to inoculate a 30 mL LB culture with the respective antibiotics (100 \u0026micro;g/mL carbenicillin for pUC19 RNR plasmids or 37.5 \u0026micro;g/mL zeocine for pREP) and grown overnight at 37 \u0026ordm;C. Plasmids were isolated from 5 mL at OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003e= 4 of each culture using the NucleoSpin Plasmid, Mini kit (Machery-Nagel). 500 ng of isolated plasmids were then digested with 20 U (1 \u0026micro;L) of both MluI-HF (NEB) and EcoRI-HF (NEB) for 1 hour at 37 \u0026ordm;C. Restriction products were verified using a D5000 ScreenTape in a 4150 TapeStation System (Agilent).\u003c/p\u003e\n\u003ch2\u003eBooting of P-FLARE in PURE reactions with TTcDR products\u003c/h2\u003e\n\u003cp\u003eTTcDR reaction products were used as input for booting P-FLARE reactions in PURE. TTcDR reactions dependent on the in vitro\u003cem\u003e\u0026nbsp;\u003c/em\u003etranslation of RNR Ia and reduction of endogenous NDPs were prepared as described above. Reactions were incubated at 30 \u0026ordm;C for 16 hours and then treated with 60 U of DpnI (NEB) for 3 hours at 37 \u0026ordm;C. P-FLARE reactions in PURE were prepared as described above, but instead of adding pET29b RNR plasmids, 2 \u0026micro;L of TTcDR reactions were added. P-FLARE reactions were then incubated for 6 hours at 30 \u0026ordm;C in a StepOne Real-Time PCR System (ThermoFisher Scientific) for 6 hours, with fluorescence measurements (EX 488 nm, EM 510 nm) every 6 minutes.\u003c/p\u003e\n\u003ch2\u003eStatistical analyses\u003c/h2\u003e\n\u003cp\u003eAll sample sizes, error bars, and statistical tests are defined in figure legends. No statistical method was used to predetermine sample size. No data were excluded from the analyses. Analyses were performed with Python (v 3.13) and Excel (v 16.100.1). Statistical significance was defined as p \u0026lt; 0.05 (* - p \u0026le; 0.05; ** - p \u0026le; 0.01; *** - p \u0026le; 0.001). When multiple comparisons were performed within the same experimental set, p values were corrected with the Benjamini-Hochberg procedure (false discovery rate \u0026ndash; FDR), with an alpha value of 0.05.\u003c/p\u003e\n\u003cp\u003eWhen different P-FLARE reactions were compared with one another, the apparent reaction rates were compared with unpaired two-tailed Welch\u0026apos;s t-tests. Comparisons were performed between apparent rates rather than maximum RFU values, since different reaction composition were observed to reach their maximum RFU values at different time points. Welch\u0026apos;s t-tests were performed as equal variance could not be assumed either due to different sample composition or due to number of measurements to assume variance normality.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen comparing transformation efficiency of different TTcDR reactions, the log-transformed CFU/mL counts of each replicate\u0026rsquo;s mean were compared to the ∆pUC-a negative control using a one-tailed two-sample unpaired Welch\u0026rsquo;s t-test to account for heteroscedasticity between samples.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorresponding authors\u003c/p\u003e\n\u003cp\u003eHannes Mutschler - Biomimetic Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, 44227, Germany; Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.M. and J.DC. conceived the project. J.DC. and N.E.N. designed experiments and collected data. J.DC. performed relevant analyses and designed the figures. V.G. purified RNR Ia subunits. All authors wrote the paper and approved the final manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Alexander Wagner (TU Dortmund University, Germany) and Deni Szokoli (TU Dortmund University, Germany) for helpful discussions and Shari L. Meichsner (TU Dortmund University, Germany) for helpful support with the purification of the RNR Ia subunits.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eForster, A. C. \u0026amp; Church, G. M. Towards synthesis of a minimal cell. \u003cem\u003eMol Syst Biol\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 45 (2006).\u003c/li\u003e\n\u003cli\u003eJewett, M. C. \u0026amp; Forster, A. C. Update on designing and building minimal cells. \u003cem\u003eCurr. 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Chem.\u003c/em\u003e \u003cstrong\u003e264\u003c/strong\u003e, 16973\u0026ndash;16976 (1989).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9248024/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9248024/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"All living organisms must maintain a balanced pool of DNA building blocks to replicate and propagate their genetic information. In living cells, ribonucleotide reductase (RNR) is the central enzyme responsible for dNTP synthesis. In contrast, minimal protein-based cell-free systems, are powerful platforms for reconstituting biological processes in vitro, but lack the endogenous metabolic pathways required to autonomously replicate their DNA and rely entirely on externally supplied dNTPs. This dependency prevents cell-free systems from achieving the metabolic autonomy required for self-sufficient genetic replication. In this work, we successfully integrated RNR’s redox activity and complex allosteric regulation for in situ synthesis of all dNTPs from endogenous NTP pools. We combined RNR’s activity to DNA synthesis and propagation of the genetic information encoding a self-contained minimal DNA replication machinery. The replication products retain the genetic information and enable re-booting of self-encoded RNR-dependent DNA synthesis. Using this strategy, cell-free systems with self-sufficient dNTP metabolism may open new avenues toward completely autonomous synthetic cells.","manuscriptTitle":"Bootstrapping DNA replication with ribonucleotide reductase in a minimal cell-free system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-08 05:02:14","doi":"10.21203/rs.3.rs-9248024/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b186da79-d8cd-45ce-9ab0-e015398ef887","owner":[],"postedDate":"April 8th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"revise","date":"2026-05-05T11:35:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-03T23:47:42+00:00","index":2,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65878513,"name":"Biological sciences/Systems biology/Synthetic biology"},{"id":65878514,"name":"Biological sciences/Biochemistry/DNA"},{"id":65878515,"name":"Biological sciences/Molecular biology/DNA replication/DNA synthesis"},{"id":65878516,"name":"Biological sciences/Molecular biology/DNA metabolism"}],"tags":[],"updatedAt":"2026-05-05T11:41:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-08 05:02:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9248024","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9248024","identity":"rs-9248024","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}