Author Response: Rolling Circle RNA Synthesis Catalyzed by RNA

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract RNA-catalyzed RNA replication is widely considered a key step in the emergence of life’s first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here, we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. We observe sustained RCS by a triplet polymerase ribozyme beyond full-length circle synthesis with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularization. Thus, all steps of a viroid-like RNA replication pathway can be catalyzed by RNA alone. Finally, we explore potential RCS mechanisms by molecular dynamics simulations, which indicate a progressive build-up of conformational strain upon RCS with destabilization of nascent strand 5′- and 3′-ends. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes. Editor's evaluation This paper is of interest to scientists from the field of origin of life or RNA synthesis in general, especially those interested in the "RNA world" scenario. The data analysis is rigorous and the conclusions are justified by the data. The key claims of the manuscript are directly related to, and support, previous findings. https://doi.org/10.7554/eLife.75186.sa0 Decision letter eLife's review process eLife digest Many organisms today rely on a trio of molecules for their survival: DNA, to store their genetic information; proteins, to conduct the biological processes required for growth or replication; and RNA, to mainly act as an intermediary between DNA and proteins. Yet, how these inanimate molecules first came together to form a living system remains unclear. Circumstantial evidence suggests that the first lifeforms relied to a much greater exrtent on RNA to conduct all necessary biological processes. There is no trace of this ‘RNA world’ today, but molecular ‘fossils’ may exist in current biology. Viroids, for example, are agents which can infect and replicate inside plant cells. They are formed of nothing but a circular strand of RNA that serves not only as genetic storage but also as ribozymes (RNA-based enzymes). Viroids need proteins from the host plant to replicate, but scientists have been able to engineer ribozymes that can copy complex RNA strands. This suggests that viroid-like replication could be achieved using only RNA. Kristoffersen et al. put this idea to the test and showed that it is possible to use RNA enzymatic activity alone to carry out all the steps of a viroid-like copying mechanism. This process included copying a viroid-like RNA circle with RNA, followed by trimming the copy to the right size and reforming the circle. These two latter steps could be carried out by a ribozyme that could itself be encoded on the RNA circle. A computer simulation indicated that RNA synthesis on the circle caused increasing tension that could ease some of the barriers to replication. These results increase our understanding of how RNA copying by RNA could be possible. This may lead to developing molecular models of a primordial RNA-based replication, which could be used to investigate early genetic systems and may have potential applications in synthetic biology. Introduction The versatility of RNA functions underpins hypotheses regarding the origin and early evolution of life. Such hypotheses of an ‘RNA world’—a primordial biology centered on RNA as the main biomolecule—are in accord with the essential role of RNA catalysis in present-day biology (Cech, 2000; Goldman and Kacar, 2021; Nissen et al., 2000; Wilkinson et al., 2020) and the discovery of multiple prebiotic synthetic pathways to several of the RNA (and DNA) nucleotides (Becker et al., 2019; Kim et al., 2020; Patel et al., 2015; Powner et al., 2009; Xu et al., 2020). In addition, progress in both non-enzymatic (Deck et al., 2011; Hassenkam et al., 2020; Prywes et al., 2016; Rajamani et al., 2008; Sosson et al., 2019; Sponer et al., 2021; Wachowius and Holliger, 2019; Zhang et al., 2020; Zhou et al., 2020) and RNA-catalyzed polymerization of RNA and some of its analogs (Attwater et al., 2018; Attwater et al., 2013; Cojocaru and Unrau, 2021; Ekland and Bartel, 1996; Horning and Joyce, 2016; Johnston et al., 2001; Mutschler et al., 2018; Shechner et al., 2009; Tagami et al., 2017; Tjhung et al., 2020) is beginning to map out a plausible path to RNA self-replication; a cornerstone of the RNA world hypothesis. RNA in vitro evolution and engineering have led to the discovery of RNA polymerase ribozymes (RPRs) able to perform templated RNA synthesis of up to ~200 nucleotides (nt) (Attwater et al., 2013), synthesizing active ribozymes including the catalytic class I ligase core (Horning and Joyce, 2016; Tjhung et al., 2020) at the heart of the most efficient RPRs, as well as initiate processive RNA synthesis using a mechanism with analogies to sigma-dependent transcription initiation (Cojocaru and Unrau, 2021). An RPR capable of utilizing trinucleotide triphosphates (triplets) as substrates (a triplet polymerase ribozyme [TPR]) has been shown to display an enhanced capacity to copy highly structured RNA templates including segments of its own sequence (Attwater et al., 2018). Nevertheless, there remain a number of fundamental obstacles to be overcome before an autonomous self-replication system can be established. A central problem among these is the so-called ‘strand separation problem,’ a form of product inhibition due to the accumulation of highly stable dead-end RNA duplexes, which cannot be dissociated (efficiently) under replication conditions (Le Vay and Mutschler, 2019). The strand separation problem has been overcome by PCR-like thermocycling (or thermophoresis) (Horning and Joyce, 2016; Salditt et al., 2020), but this approach may be limited to short RNA oligomers (even in the presence of high concentrations of denaturing agents) as the melting temperatures of longer RNA duplexes approach or even exceed the boiling point of water (Freier et al., 1986; Szostak, 2012). While RNA duplexes occur by necessity as intermediates of RNA replication, the extent of the strand separation problem can be modulated by genome topology. Circular rather than linear genomes are widespread in biology including eukaryotes, prokaryotes, and viruses (Moller et al., 2018; Moss et al., 2020; Shulman and Davidson, 2017). Circular RNAs (circRNAs) are found as products of RNA splicing (Kristensen et al., 2019) and RNA-based self-circularization is known in multiple ribozymes (Hieronymus and Müller, 2019; Lasda and Parker, 2014; Petkovic and Müller, 2015). Continuous templated RNA synthesis on circular templates (rolling circle synthesis [RCS]) is also widespread and found in the replication of the RNA genomes in some viruses and in viroids. Indeed, viroid RNA replication has been proposed to resemble an ancient mechanism for replication (Diener, 1989; Flores et al., 2014). In an idealized RCS mechanism, both strand invasion and displacement processes are isoenergetic and coordinated to nascent strand extension (Blanco et al., 1989; Daubendiek et al., 1995), with rotation of the single-stranded RNA (ssRNA) alleviating the build-up of topological tension (Kuhn et al., 2002). Thus, RCS is a potentially open-ended process leading to the synthesis of single-stranded multiple repeat products (concatemers) with an internally energized strand displacement (Tupper and Higgs, 2021). RCS as a replication mode has therefore potentially unique properties with regards to the strand separation problem. Specifically in the context of triplet-based RNA replication on a circular template, duplex dissociation, and strand separation may in principle be driven by trinucleotide (triplet) hybridization and ligation, leading to extension of the nascent strand 3′-end and an equal displacement of the 5′-end in triplet increments (Figure 1A). Triplet binding to the template strand and dissociation of an equal trinucleotide stretch from the 5′-end are both equilibrium processes and nearly isoenergetic. However, extension (i.e., ligation of the bound triplet to the growing 3′-end) is an irreversible step. Thus, in this scenario, RCS would be expected to proceed in ratchet-like fashion with strand displacement driven by triphosphate hydrolysis and triplet ligation. Figure 1 with 1 supplement see all Download asset Open asset Primer extension on circular RNA (circRNA) templates. (A) Schematic illustration of rolling circle synthesis (RCS). Red product RNA strand is extended by a triplet at the 3′-end while three base pairs dissociate at the 5′-end keeping the total hybridization energy constant. Topological relaxation is allowed by rotation of the single stranded part of the circular template (swiveling arrow). (B) Linear or circularized RNA is treated with or without endo- or exonucleases (RNase A/T1 mix or Exonuclease T (ExoT)). Only circRNA is resistant to ExoT digestion. (C) Schematic representation of primer triplet extension on a linear or circRNA template and of the TPR hetero-dimer comprising the catalytic subunit (5TU (green)) and the non-catalytic subunit (t1 (red)) (Attwater et al., 2018) (TPR sequence: Supplementary file 1). (D) PAGE gel of TPR primer extension, P9 (5′-FAM-GAAGAAGAA) is the unextended primer, bands 1–9 denote extension of P9 by +1 to + 9 triplets (full length). RNA template used was sc12GAA-p (36 nt, 12 repeat UUC template). Experiment was done under standard conditions in the Tris buffer system described in Materials and Methods except that 800 pmol triplets were used. (E) Extension efficiency of formation of bands 1–9 in (D) (see Materials and methods) is plotted against triplet position. (F) Schematic model of the sc12GAA-p illustrating the different accessibility of in- or outside facing triplet junctions on the scRNA template (blue), with P9 primer (red), and the product strand (light gray). Original gel images and numerical values are supplied in Figure 1—source data 1. Figure 1—source data 1 Gel images and numerical values. https://cdn.elifesciences.org/articles/75186/elife-75186-fig1-data1-v3.zip Download elife-75186-fig1-data1-v3.zip Here, we have explored triplet-based RCS of scRNA templates as a potential solution to the strand separation problem in RNA-catalyzed RNA replication. We show that RCS can be catalyzed by the triplet polymerase ribozyme [TPR] (Attwater et al., 2018). We show that the TPR is able to perform continuous templated extension of circular RNA templates beyond full-length circle synthesis with strand displacement yielding concatemeric RNA products. We also investigated the mechanistic basis for RCS and strand displacement by molecular dynamics (MD) simulations of scRNA in explicit solvent. Finally, we consider the potential of a full viroid-like replication cycle catalyzed by RNA alone by design and synthesis of a circular Hammerhead ribozyme capable of both product cleavage and self-circularization. Results RNA-catalyzed primer extension using small circular RNA templates We first set out to investigate whether templated RNA synthesis on scRNAs as templates could be catalyzed by an RNA catalyst. To extend the RNA nascent strand beyond the full circle and initiate RCS requires duplex invasion and displacement of the original RNA primer and product strand. However, most RPRs are inhibited by duplex RNA both in the form of template secondary structures and as linear duplex RNA. We therefore explored the potential of a recently described TPR (Attwater et al., 2018), which is able to utilize trinucleotide triphosphates (triplets (pppNNN)) as substrates for polymerization. Due to increased binding of the triplets to the template (compared, e.g., to the canonical mononucleotide triphosphates (pppN, NTPs)), triplets are able to invade and cooperatively ‘open up’ template secondary structures for replication (Attwater et al., 2018). We hypothesized that this ability might also promote the continuous invasion and displacement of the nascent strand 5′-end and facilitate the RCS mechanism (Figure 1A). As described previously, RNA synthesis by the TPR is most efficient in the eutectic phase of water ice, due to its beneficial reaction conditions for ribozyme catalysis (Attwater et al., 2018). Specifically, eutectic ice phases aid TPR activity by the reduced degree of RNA hydrolysis under low-temperature conditions, reduced water activity, and the high concentrations of reactants (ribozyme, scRNA template, triplet substrates, and Mg2+ ions) present in the eutectic brine phase that arises by excluding solutes from growing water ice crystals and remains liquid at subzero temperatures (Attwater et al., 2010). Thus, all RCS experiments were carried out under eutectic conditions. We prepared scRNA templates (34–58 nt in length) by in vitro transcription and ligation. Circularity of the scRNA templates was verified based on gel mobility and resistance to exonuclease (exoT) degradation in contrast to the linear, non-ligated counterparts (Figure 1B, Figure 1—figure supplement 1/sequences for all oligonucleotides in Supplementary file 1). We first investigated primer extensions using a single triplet (pppGAA) on the scRNA template as this provides an even banding pattern of incorporation. This facilitates analysis and allows primer extension efficiencies of linear and circular templates to be more readily compared (Figure 1C). Primer extension experiments using a purified 36 nt scRNA as template resulted in full-length extension around the circle (Figure 1D), but with reduced efficiency compared to a linear RNA template. Furthermore, we observed a periodic banding pattern of triplet extension efficiency matching the helical pitch of double-stranded RNA (dsRNA) (11.3 base pairs (bp)/turn Bhattacharyya et al., 1990; Figure 1E). Presumably, triplet junctions located on the inside of the scRNA ring are less easily accessible and therefore less efficiently ligated than in linear RNA, which is freely accessible from all sides (Figure 1F). This may explain both the observed periodicity and reduced synthetic efficiency of RNA synthesis by the TPR on scRNAs. Despite the reduced extension efficiency in scRNA, we obtained full circle extension products for multiple templates (34–58 nt in size, Figure 2A and B) with a clear trend toward increasing mean extension efficiency for circular templates with increasing size predicting parity with the linear template at around 120 nt (Figure 2C). Note, in these experiments extension beyond full circle was not intended or possible (lanes 1–6 in Figure 2B) as the specific triplet substrates needed for displacing the primer were not present in the reaction. Figure 2 with 1 supplement see all Download asset Open asset Full-length and beyond full-length RNA-catalyzed RNA synthesis on circular RNA templates. (A) Product strand of primer extension experiments with primer P10 (red) and eight triplet scRNA template strands. Potential beyond full-length circle synthesis triplets are shaded opaque. (B) Various scRNA template sizes allow full-length primer extension as indicated (with eight triplet sites) (gray), GAA triplets (black), and primers P10 (5′-FAM-CUGCCAACCG) or P10 +3 (5′-FAM-GAAGAAGAA-CUGCCAACCG) (red). PAGE of primer extensions (under standard conditions) with full-length synthesis for different scRNA templates (34–58, termed scGAA8-16, Supplementary file 1) marked by a black line. (C) Mean extension efficiency plotted as a function of scRNA size calculated from extension experiments including (B) (error bars indicate standard deviation, n=5), with mean extension efficiency for a linear RNA template (red dashed line). (D) PAGE of time-course of primer extension of primer P9 on scRNA template, sc12GAA-p (optimized conditions). Full-length circle synthesis is marked by a dashed black line (after +9). Bands +10 and beyond (see Enhanced contrast section) indicate beyond full-length synthesis. (E, F) Mean extension efficiency (from (D)) plotted against time (E) or triplet position (F), showing the respective amounts of product at full-length (black) and beyond full-length circle (red) synthesis as well as the drop in efficiency at full length, which recovers once beyond full-length synthesis is initiated. Vext and Vinv denote the calculated velocity of formation of bands 9 and 10, respectively. Original gel images and numerical values are supplied in Figure 2—source data 1. Figure 2—source data 1 Gel images and numeric values. https://cdn.elifesciences.org/articles/75186/elife-75186-fig2-data1-v3.zip Download elife-75186-fig2-data1-v3.zip Having established full-length synthesis on scRNA templates, we next tested if primer extension could proceed beyond full circle requiring duplex invasion and displacement of the primer/product strand. We first tested this using primer P10 +3, comprising a 5′ extension of three GAA repeats, thus covering the last three UUC triplet binding sites on the circular template (Figure 2B, top right). We observed an extension of up to +3 triplets (+9) above the full circle mark (Figure 2B, lanes 7–12), indicating displacement of the primer 5′-end upon incorporation of three additional pppGAA triplets. This indicated that synthesis beyond full circle including strand displacement is possible on scRNA templates, boding well for the implementation of RCS. To that effect, we next optimized buffer and extension conditions for more efficient extension above the full circle mark (Figure 2—figure supplement 1). Interestingly, greater dilution of reaction mixtures prior to freezing resulted in more efficient stand displacement. While greater pre-freezing dilution does not alter the final solute concentrations within the eutectic phase (Attwater et al., 2010), it increases the eutectic phase/ice interface area. This suggests that strand invasion may be aided by surface effects, as previously suggested for RNA refolding (Mutschler et al., 2015). Under these optimized buffer and extension conditions, we observed progressive accumulation of progressively longer RCS products, over prolonged reaction times (up to 6 weeks) (Figure 2D) with reaction speed decreasing after ca. 4 weeks incubation, indicating continued RCS over extended periods of time (Figure 2E and F). Molecular dynamics simulations of 36 nt scRNA To better understand the structural and topological constraints of RCS on scRNAs, we performed atomistic MD simulations over 400 ns of the different RCS stages, comprising the starting scRNA template as circular ssRNA and scRNA with a progressively extended dsRNA segments (Figure 3). For simplicity, a 36 nt circular RNA sequence of (UUC)12 was chosen as a template strand (similar to the scRNA template [sc12GAA-p] in Figures 1 and 2D) for direct comparison with the experimental system. The complementary strand comprising GAA triplets starting from 9 bp dsRNA (corresponding to binding of primer P9) was extended from 18 to 30 bp (after +3 triplet incorporation) in triplet increments of dsRNA corresponding to extension products of bands +3, +4, +5, +6, and +7 (see gel in Figure 1D), using the most representative structure of the previous simulation as a starting point for the next one. Figure 3 with 6 supplements see all Download asset Open asset Molecular dynamics simulation of small circular RNA. (A) Main conformations (and zoom-in to relevant regions [squares]) observed from simulations in 100 mM MgCl2 on scRNA exploring consecutive states of primer extension, from 9 to 30 bp dsRNA with pyrimidine (template) strand (UUC)12 (khaki), purine (product) strand (GAA) (light blue), 5′-end and unpaired bases (dark blue) and 3′-end unpaired bases (purple) and matching melted bases from the template strand (dark green (5′-end)/light green (3′-end)). (B) Percentage of frames from the last 100 ns of the simulations presenting canonical hydrogen bond pairing for each bp. (C) Counterion-density maps (in red) around RNA molecules that show an occupancy ~10 times or greater than the bulk concentration. The simulation trajectories revealed the high energy barrier of dsRNA for bending and accommodating a circular shape (Figure 3A). Instead, we observe that, as dsRNA is elongated, the remaining ssRNA segment of the scRNA becomes increasingly extended. As the dsRNA part reaches 27 bp (corresponding to band 6 in Figure 1D), the ssRNA segment was fully extended and torsional strain was relieved by dissociation (peeling off) of the dsRNA 5′- and 3′-ends rather than by bending or the introduction of kinks into the dsRNA segment (Figure 3B). Subsequently, multiple peeling off and rebinding events were observed during the trajectories indicating that the dynamics of this process are fast (Videos 1 and 2 and Figure 3—figure supplements 1 and 2). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Movie of the RCS simulation where dsRNA is 27-bp long. We observe fraying and annealing of 5′- and 3′-ends demonstrating the quick timescales of these transitions. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Movie of the RCS simulation where dsRNA is 30-bp long. We observe again fraying and annealing of 5′- and 3′-ends demonstrating the quick timescales of these transitions. In the experimental data, we also observed an inhibitory effect for insertion of the final triplets (+8, +9, and +10 (beyond full length)/extension to 33, 36, and 39 nt of RNA in Figure 2D) into the corresponding scRNA template. This may indeed reflect the onset of the 3′- and 5′-ends destabilization observed in the MD simulations (Figure 3), which would likely attenuate primer extension by the ribozyme. Note however that the extension efficiency recovered beyond full length (+11/extension to 41 nt, Figure 2F), although at lower speed (Figure 2E). As a control for the observed dsRNA end destabilization mechanism, we also ran an MD simulation of a linear RNA molecule containing four consecutive triplets with a nick between two of them, but observed neither base opening nor dissociation at either 5′- or 3′-strand ends (Figure 3—figure supplement 3). Groove dimensions and local helical parameters (roll, twist, and slide) for the RCS simulations on circular RNA did not show any major adjustment compared with the linear RNA control (Figure 3—figure supplement 4). We observed an oscillation of high/low values of bending along the molecule in phase with RNA-turn periodicity in an attempt to create an overall curvature (Velasco-Berrelleza et al., 2020), although with moderate success (~60° on an arc length of 30 bp of dsRNA) and no formation of kinks, internal loops or other disruption of the canonical A-form typical of the RNA duplex (Figure 3—figure supplement 4). To mirror the experimental eutectic phase conditions, simulations were run at relatively high Mg2+ concentrations (100 mM) and compared with the presence of monovalent ions like K+ (200 mM) and high concentration of Mg2+ (500 mM), but simulations did not show any major differences in terms of dsRNA strand dissociation or bending (Figure 3—figure supplements 3 and 4). However, Mg2+ ions—compared to K+—appear to interact more strongly with different parts of the RNA and, consequently, may increase the probability of distorted conformations facilitating the exposition of nucleobases at the 5′- and 3′-ends. In contrast, K+ ions are mainly positioned as counterions along the major and minor groove, allowing the bases to orient toward the inside of the dsRNA helix for base-pairing interactions (Figure 3C and Figure 3—figure supplement 5). The role of Mg2+ in the stabilization of complex RNA folding has been observed repeatedly in several structures (Sponer et al., 2018), like the ribosome (Klein et al., 2004) and the Hepatitis delta virus ribozyme (Nakano et al., 2001). However, increasing MgCl2 concentration to 500 mM does not seem to bring extra stabilization, as the system appears to be saturated already at 100 mM Mg2+ (Figure 3—figure supplements 5 and 6). In summary, our simulations support the notion that progressive RNA synthesis on a scRNA template (in the presence of Mg2+ ions) leads to increased dynamics of nucleobase exposure, RNA duplex destabilization, and 5′- and 3′-ends melting. The simulations also clearly illustrate the implausibility of a small circular fully dsRNA molecule (as schematically illustrated in Figure 1E), due to the prohibitive energetic cost of dsRNA bending. Instead, the system appears to relieve internal strain by extending the ssRNA segment of the circle (partially shielding the dsRNA segment) and peeling of both dsRNA 5′- and 3′-ends (Figure 3), consistent with the helical period of triplet extension observed (Figures 1 and 2) (with ligation junctions facing into the ssRNA center being less accessible) and the observed reduction in RCS efficiency. Dynamic destabilization of dsRNA 5′-ends clearly has the potential to facilitate strand displacement during RNA replication on a scRNA template (and may aid continuous RCS), but at the same time may reduce the efficiency of primer extension and triplet incorporation by reducing the availability of the primer 3′-end and the downstream template bases. These effects would be predicted to manifest themselves in RNA circles up to 200 bp as suggested by RNA persistence length (Abels et al., 2005). Templated rolling circle RNA synthesis Having validated RNA synthesis on scRNA templates (Figures 1 and 2), we next sought to show RCS beyond a single full-length circle synthesis involving displacement of the primer/nascent strand. To this end, we designed barcoded templates that would allow us to distinguish RNA synthesis products arising from template-instructed RCS from those arising from non-templated terminal transferase (TT) activity of the TPR by sequencing. The barcoded scRNA templates (termed A–D) were prepared either as circular or linear RNAs comprising different internal triplet ‘barcodes’ of variable GC-content (at positions 3, 6, and 9) for individual identification (Figure 4A and Figure 4—figure supplement 1). We performed one-pot primer extension experiments, in which all four templates (either A–D linear or A–D circular) were mixed in equal proportions. After extension, products were separated by gel electrophoresis and the gel sections above full-length extension products were excised and their RNA content recovered, and sequenced (Figure 4—figure supplement 1). Diagram in Figure 4B represents (in %) which triplets were identified at the noted positions. Boxes mark expected triplet according to the template sequence. Figure 4 with 2 supplements see all Download asset Open asset RNA-catalyzed RNA synthesis beyond full length for circular templates. (A) Product strands of primer extension experiments with linear and scRNA templates A–D with primer P91. Opaque sequence illustrate potential beyond full-length synthesis on scRNA. Barcode triplets at positions 3 (A/U rich) (cyan), 6 (mix) (blue), and 9 (G/C rich) (purple) allow identification of product RNAs. Barcode triplet at position 12 is the final triplet of primer P91 and at position 15 is the same as that of position 3 but after beyond full-length circle synthesis on the scRNA template. (B) Fidelity heatmap of the sequences derived from the one-pot experiments with linear (left) or circular (right) templates. Red color indicates high prevalence of a given triplet (vertical axis) at the position noted (3–18). n: denotes the number of sequence reads (3E5=3×105) used to calculate the fidelity for each triplet at the given position. Transparent gray boxes cover positions with n≤5. (C) Plot shows ratio (fold difference) of the probability of a product of reaching positions 4–12 on circular compared to linear templates. Fold difference was calculated based on fidelity data presented in (B). (D) Model illustrating (1.) beyond full-length extension on a circular template (templated RCS) and (2.) on a linear template (non-templated). Full analysis of the data in B is supplied in Figure 4—source data 1. Figure 4—source data 1 Full analysis of sequencing data used in Figure 4B. https://cdn.elifesciences.org/articles/75186/elife-75186-fig4-data1-v3.zip Download elife-75186-fig4-data1-v3.zip Analysis of the sequencing products from the one-pot reaction showed template-dependent high-fidelity RNA synthesis up to full length (position 9) for all templates (linear and circular) (Figure 4B). Further, all templates yielded longer than full-length products indicative of continued RNA synthesis by the TPR beyond full length (positions>9). However, the fidelity dropped after full length was reached, indicative of significant non-templated terminal transferase-like (TT) activity in this regime (Figure 4B). For example, the average fidelity for insertion of the expected triplet (pppGAA) for position 10 (full length +1) for circular templates was 10.9% whereas for position 9 (full length) it was 89.9%. For linear templat