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. . Yet, how these inanimate molecules first came together to form a living system remains unclear.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 proteinsCircumstantial 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. 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  and the 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) , synthesNevertheless, 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 . The strWhile 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 . CirculaIn an idealized RCS mechanism, both strand invasion and displacement processes are isoenergetic and coordinated to nascent strand extension , with roHere, 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] . We showpppNNN)) as substrates for polymerization. Due to increased binding of the triplets to the template ), triplets are able to invade and cooperatively ‘open up’ template secondary structures for replication  present in the eutectic brine phase that arises by excluding solutes from growing water ice crystals and remains liquid at subzero temperatures  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 (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 . We firscompared . Primer compared , but witcompared . Presumacompared . This maDespite the reduced extension efficiency in scRNA, we obtained full circle extension products for multiple templates /extension to 33, 36, and 39 nt of RNA in 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 . Groove 2+ 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  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  for position 10 (full length +1) for circular templates was 10.9% whereas for position 9 (full length) it was 89.9%. For linear templates, the fidelity for full length +1 dropped to 0.7% compared to full length 78.8%. Note, that fidelity at full length +1 dropped much more for linear than for circular templates. For this reason, the probability of a product extending to longer than full length (positions 10–12) with the correct sequence was 10–100-fold higher for circular compared to linear RNA templates  for all templates (linear and circular) . Furtheremplates . A few eemplates  were alsOn all circular templates  extension beyond full length (while containing a significant TT component) continued to insert the barcode triplets correctly, indicating continuous RCS at least up to position 18 . Control experiments, with individually incubated templates (in contrast to the one-pot experiments) mixed after gel purification, showed essentially identical results . InterespppGAA triplet was present, primers were extended only by two triplets as expected  were present, extension continued to beyond full circle (positions≥10)   and bandions≥10) .3 and 1×105 reads of products I and II, respectively). However, much longer sequences were present in decreasing numbers of reads, with the longest products comprising 29 correct triplet incorporations (96 nt) (n=2) representing RCS of >2.5 times full-length circle synthesis and the longest reported product synthesised by the TPR. Thus, RNA-catalyzed RCS has the potential to yield extended RNA concatemer products under isothermal conditions. Freeze-thaw (FT) cycles have been previously shown to enhance ribozyme activity by affecting RNA refolding  of the concatemer into individual units and circularization of unit length RNAs by ligation to recreate the original circular RNA genome. As both RNA cleavage and RNA ligation can be efficiently catalyzed by RNA, we sought to investigate, if a viroid-like replication cycle might be catalyzed by RNA alone. To this end, we designed a proto-viroid RNA comprising a 39-nt scRNA template encoding a designed micro-hammerhead ribozyme (µHHz) as well as its substrate for cleavage . We enviWhen the µHHz had been 5′-phosphorylated , lane 3 Avsunviroidae encoding a Hammerhead ribozyme responsible for maturation by resecting the replicated RNA genome  and re-circularized by an encoded ribozyme in a scheme reminiscent of viroid replication. Thus, one-half of a viroid replication cycle ((−)-strand replication leading to a self-circularizing (+)-strand) can be carried out by just two ribozymes. Completing a full viroid replication cycle would then require the reverse (+)-strand replication leading to a self-circularizing (−)-strand product. This may require a second ribozyme  encoded in the (−)-strand akin to the mechanism used by natural viroids .MD simulations indicate that the RCS process may be aided by accumulating strain in the nascent dsRNA segment leading to increased displacement (peeling off) of dsRNA 5′- and 3′-ends . In turn, this peeling off creates a more dynamic environment potentially aiding 5′-end invasion by extending the 3′-end. This topological strain-induced strand displacement may be general and independent of the precise RCS mechanism on scRNA templates and thus should also apply to non-enzymatic polymerization of RNA. Our observation that RCS can be enhanced by the use of branched extension, FT thermocycling, and pre-freezing dilution may also be related to this. Indeed, while the precise mechanistic basis for these enhancements is currently unknown, it seems plausible that all of these enhance 5′-end displacement on the product strand by accelerating conformational equilibration through RNA un- and refolding as observed previously .In biology, both viroids and Hepatitis D virus (HDV) replication proceeds through RCS on circular RNA genomes mediated by proteinaceous RNA polymerases, but RCS has also been reported for circular DNA templates and proteinaceous DNA polymerases in nature  and in bLarger circular RNA templates might provide advantages for the RCS as they are less strained. They would also provide increased access to the internal face of the circle and might be able to encode the whole ribozyme itself. On the other hand, reduced torsional strain on the dsRNA would be expected to reduce strand invasion and ‘peeling off’ of the product strand. All of these factors merit detailed investigation.Our motivation for investigating RNA-catalyzed RCS was as a potential solution toward the so-called ‘strand separation problem,’ the inhibition of RNA replication by exceedingly stable RNA duplex products. This form of product inhibition has both a thermodynamic component, that is, the high amount of energy required to dissociate RNA duplexes, and a kinetic component as RNA replication must outpace duplex reannealing, which is rapid unless duplex concentrations are low or reactions take place in a highly viscous medium . In thisIn the course of this work, we discovered another property of RNA synthesis on circular RNA templates that might contribute toward overcoming the strand separation problem. MD simulations indicate that—at least—on scRNA templates—the build-up of strain in the nascent dsRNA segment could aid strand displacement . HoweverBase sequences of all oligonucleotides used throughout this work can be found in In vitro transcription dsDNA templates (containing T7 promotor sequence at the 5′-end upstream of the region to transcribe) for in vitro transcription was generated by ‘fill-in’ using three cycles of mutual extension using GoTaq HotStart  between the relevant oligonucleotide and primers: 5T7 or HDVrt (the latter for defined 3′ terminus formation The sample in the appropriate loading buffer was separated on 10%–20% 8 M Urea denaturing PAGE gel using an EV400 DNA Sequencing Unit (Cambridge Electrophoresis). The product band was visualized by UV shadowing (for non-labeled RNA) or fluorescence scanning  (for labeled RNA). When needed the identified product based on relative migration was excised. The excised gel fragment was then thoroughly crushed using a pipette tip and suspended in 10 mM Tris-Cl pH 7.4 to form a slurry. For freeze and squeeze extraction, the slurry was frozen in dry ice, then heated to 50°C (~5 min), and finally left rotating at room temperature (from 2 hr to overnight) to elute the product from the gel material. The eluate was then filtered using a Spin-X column , ethanol precipitated, (100 mM Acidic acid and 80% ethanol (10 μg glycogen carrier was present when noted)). UV absorbance was measured with a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific) to determine yield of redissolved purified RNA.Gel images from the Typhoon scanner were analyzed in ImageQuant software (Cytiva Life Science) for quantifying band intensity. Quantified band intensities were exported to Excel  for further analysis. Extension efficiency (E) for a given band (b) was calculated as the sum of the intensities (I) of all the bands from b to n (n being the highest detectable band), divided by the sum of I of all bands from b−1 to n:Thus, E represents the efficiency of the given ligation junction (Lb) to allow the production of the extension product in band b, that is, the extension efficiency.Triplets were prepared via run-off in vitro transcription with T7 RNA polymerase. More details on the method can be found in Linear 5’-end monophosphate labelled RNA to be used for circularization was either prepared by in vitro transcription (300 μl reaction volume) or ordered directly as chemically synthesized RNA . Linear RNA was gel purified as described above. When needed purified RNA was treated with T4 polynucleotide kinase (PNK) (New England Biolabs (NEB), Ipswich, MA) to remove 3′-end cyclic phosphate then RNA was phenol/chloroform extracted, ethanol precipitated, and redissolved in ddH2O. For splinted ligation, 3 pmol purified RNA was mixed with equimolar splint RNA in 262.5 μl ddH2O. The sample was heated to 80°C (2 min) followed by cooling to 17°C (10 min) and finally incubated on ice (5–30 min). Then reaction conditions were adjusted to 50 mM Tris-HCl pH 7.5, 2 mM MgCl2, 1 mM DTT, and 400 μM ATP (1× T4 RNA ligase 2 reaction buffer [NEB]) including 0.25 units/μl T4 RNA ligase 2 (Neb)  and samples left overnight (~16 hr) at 4°C. For non-splinted ligation, 0 pmol gel purified RNA was mixed in 237 μl ddH2O followed by heating to 95°C and then quickly moved to ice. Then reaction conditions were adjusted to 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT (1× T4 RNA ligase reaction buffer [Neb]), 100 μM ATP including one unit/μl T4 RNA ligase 1 (NEB)  and samples left overnight (~16 hr) at 16°C. Circularized RNA was electrophorated by 10% 8 M Urea denaturing PAGE for analysis and purification as described above.Ribozyme activity assay was performed essentially as described in In the primer extension reactions used for sequencing, the primer extension was performed as described above except for the following changes: 5 pmol ribozyme heterodimer/template, 20 pmol primer (with a 5′ adapter sequence), and 100 pmol of each triplet was used. In the cases where multiple templates were mixed in the same reaction (one-pot), the final template concentration remained 5 pmol in total. All reactions were done in the CHES buffer and were diluted 50× as standard.After Urea PAGE separation of the extension products, the noted region of the gel was dissected out, and carefully recovered as described above. The RNA was ethanol precipitated (80% ethanol with 10 µg glycogen carrier) resulting in a dry RNA pellet. To append an adaptor sequence to the 3′-end of the purified RNA products the dry RNA was redissolved in conditions allowing adenylated adapter ligation by T4 RNA ligase 2 truncated K227Q (Neb) following manufacturers’ descriptions. Final adapter ligation conditions were: 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 1 mM DTT (1× T4 RNA ligase reaction buffer [NEB]), 15% PEG8000, 0.04% Tween 20, 5 pmol adenylated DNA primer  as described by the manufacturer and transformed into heat-competent 10-Beta cells (NEB). Inserts from single colonies were PCR amplified (using primers pGEM_T7_Fo and pGEM_SP6_Ba) and send in for Sanger sequencing (Source Bioscience) (using pGEM_T7_Fo as sequencing primer).x_Fo, x denotes different barcodes 1–15, see oligo sequences in Illumina adaptors were added to purified RT-PCR products by PCR (15 cycles) using 0.5 µM forward (Ill1) primer sequence (GAAGAACTG). After the P91 sequence, the triplets at positions 1, 2, 3, and so on, would be identifiable representing extension products made by the ribozyme. The presence of the 3′ adapter sequence (GTCGAATAT…) in the aligned sequences marked the end of the original RNA extension product. Sequencing data can be found as described below under section data availability: File 1 includes sequence data for circular and linear one-pot analysis , File 2 includes sequence data for branched RCS analysis (B3).Illumina Sequencing data were acquired and processed as FASTQ files using Terminal (and available software packages such as FASTX-toolkit). Prior to analysis, the whole output file from Illumina sequencing runs  was split based on barcodes identifying the individual samples and trimmed starting with the original  with a list of all relevant sequences (positions 3–18) and the sequencing files. The triplet at position 3, the first barcode position, was used to classify the sequences into coming from templates A to D and thus has 100% fidelity for the correct triplet  for each position was done using  triplet .(primer)GAA(pos1)GAA(pos2)YYY(pos3)XXX(pos4). Here, YYY was either of the first barcode triplets for templates A–D, (ATA(template A), AAA(template B), TTA(template C), or ATC(template D)) and XXX was either of the 14 possible triplets . F at position 4 was then calculated for templates A–D as the number of occurrences of a triplet in position 4  divided by the sum of occurrences of all the triplets multiplied by 100%. A generalized term for calculating the F at all positions (3–18) and for all templates (A–D) is:For example, for analyzing the fidelity (F) of position 4, the following list was used: GAAGAACTGHere, F is the fidelity, a is the position of the triplet, Y is the templates A–D. n is the number of sequencing reads for a given triplet (xxx) for position a on template Y or for all the 14 triplets (XXX), for a on Y. Eventually, the fidelity for positions 3–15 in the context of templates A–D for all triplets was plotted in grep  with a list of all relevant sequences , and the sequencing file. Data for this analysis can be found as described in the Data availability section below (File 2). Numerical data and calculation are supplied in By counting the number (n) of correct sequences with a specific length ending in the 3′ adapter sequence, we identified long RCS products . This waRNA-catalyzed synthesis of fluorophore labeled self-circularizing µHHz was prepared in 2× large (500 pmol) reactions set up and incubated as described above. Specifically, 500 pmol ribozyme heterodimer (5 TU/t1) and circular template (scHHz_temp), 2000 pmol primer (HHrzP12) and 50 µmol of each of the triplets were annealed followed by adding buffer to 50 mM CHES, pH 9, 150 mM KCl, 10 mM MgCl2, and 0.05% Tween 20 (1 ml). Then the sample was diluted 50 times to a final volume of 50 ml. After 4 weeks incubation at –7°C, EDTA was added , reactions were thawed and concentrated to a final volume of ~300 µl using a centrifugation filter  retaining long RNA products. µHHz RNA  was gel purified as described above and excised product was dissolved to 10 µM in H2O with 0.5 mM EDTA.µHHz annealed  in 4 µl water with 1 µl 5× reaction buffer, final reaction conditions: 50 mM CHES, pH 9, 150 mM KCl, and 10 mM MgCl2 . Then incubated in ice for 5 min to ensure folding. This was then frozen on dry ice and either moved to –7°C for eutectic phase formation (reaction) or –80°C (control). After incubation, 10 µl loading buffer  was added directly to the cold samples to stop the reaction and mixed while thawing. Finally, reactions were analyzed by 20% denaturing PAGE like described above. 5′-phosphorylation of µHHz RNA with polynucleotide kinase (NEB) was carried out following the manufacturer’s directions. RNA was then phenol/chloroform extracted, precipitated, and dissolved in ddH2O with 0.5 mM EDTA to 10 µM (determined by Nanodrop).Self-circularization assays of chemically synthesized fluorophore labeled µHHz comprise 10 pmol 12 was built using the NAB utility, which was then circularised using an in-house program  set of ion parameters (2. Simulations were performed at constant T and P (300K and 1 atm) following standard protocols .Thank you for submitting your article "Rolling Circle RNA Synthesis Catalysed by RNA" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Reviewer #1:In the origin of life scenario where RNA is assumed to be the first replicator, a key problem is how RNA can replicate itself. Or how can RNA polymerase copy itself, since copying requires an open flexible structure that can be read. While the polymerase needs to be a topological rigid structure in order to catalyse the RNA polymer.The manuscript describes how a special kind of synthesis of the RNA, rolling circle synthesis on small pieces of circular RNA, could template and build RNA strands. The method apparently could help in avoiding the strand inhibition problem where stable RNA duplex in long strands hinders replication.It is a bit unclear how, but I suspect this i down to the size of the ring, since the problem increase with the length of the polymer. The authors also touch on this themselves in their MD simulations, since the entropically unfavourable confinement of a string onto a circle can alleviate part of the problem. Nevertheless, the authors also show by MD that the rings become small tight structures that actually hinders replication.The study depends on trinucleotide triphosphates (triplets) as substrates. They demonstrate a viroid like replication that show how a template (-) is able to make a mirror copy (+) of circular RNA by a rolling circle synthesis with out the need for enzymes or other catalyst apart from RNA itself.While the conclusion of the work becomes a bit muddled, but honest, the work is a very important piece that demonstrates the huge potential role of circular RNA in the very early stages of life.I must confess I am very far off my field of competences. I have tried my best to understand the paper, and the methods involved, but obviously I cannot give much feedback on the methods used. So, I cannot suggest improvements on that part.For the most part, the paper is well written, and the structure is sound. I would strongly recommend publication.It was unclear to me how it was certain that it was actually rings. There is the MD simulations, but apart from the principle drawing they are obviously not circular in shape.There are some parts of the manuscript that too me is very difficult to understand. Some statements seem to reflect a community understanding that might be obvious for those working with similar system on a day to day basis, but for the general reader (where I put myself) this becomes gibberish. for instance:RCS has potentially unique properties with regards to the strand inhibition problem where RNA duplex melting in principle can be effected by continuous toehold strand displacement driven by nucleotide hybridization and the ratchet of nascent strand extension by triphosphate hydrolysis. In an idealized RCS mechanism, such strand invasion and displacement processes are both isoenergetic and coordinated to nascent strand extension , with rotation of the single-stranded RNA (ssRNA) preventing the build-up of topological tension . 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 circumventing the "strand inhibition problem" .It might not be possible to rephrase this so that everyone can understand. But I think the clarity of manuscript could be improved.But also because there is references to previous work where long statements does not make it more clear what is actually meant. for instance:Similar to what was described previously, RNA synthesis by the TPR best in the eutectic phase of water ice, due to beneficial reaction conditions for ribozyme catalysis such as reduced RNA hydrolysis and high ionic and RNA substrate concentrations . This was also the case on scRNA templates.That said, I think the overall message is clear and the paper is very interesting, I expect to reference it when it comes out.Reviewer #2:The RNA World theory is one of the most widely-believed explanations for the origin of life. This relies on the idea that there were self-replicating RNA systems in the early stages of life. Usually ,it is supposed that there were polymerase ribozymes that were able to use another RNA strand as a template for synthesis of the complementary strand. As there are no naturally-occurring polymerase ribozymes, there has been a sustained effort over several decades to develop polymerase ribozmes in the lab by in vitro selection. This paper contributes to this by presenting a polymerase ribozyme that can copy a circular template. Circular templates are thought to be important because replication of a circular template can occur via the rolling circle mechanism, in which a polymerase continues multiple times around the same circle, and the far end of the growing strand is displaced from the template at the same time as new bases are added to the growing end. This avoids the problem of strand inhibition (i.e the difficulty of separation of stable double strands that are expected to form when copying linear templates).This paper considers rolling circle replication on very short circles of around 36 nucleotides. It is shown that replication proceeds by addition of triplets beyond the full length of the circle. As the circle is short, and the double-stranded part is stiff, it is not possible for the whole of the circular template to be double-stranded at the same time. It is shown that roughly half of the circle is double-stranded, and that the separation of the two strands occurs at a point which is on the opposite side of the circle from the point of primer extension.The rolling circle mechanism involves cleavage of the growing strand by a self-cleaving hammerhead ribozyme that is encoded in its sequence. The mechanism also requires the reconnection of the ends of the new strand in order to form a new circular template. Both the cleavage and re-circularization steps are demonstrated in this paper.This experiment still falls short of a fully self-replicating ribozyme system, because in order for continued replication to occur, both plus and minus strands of the circle would have to encode a hammerhead ribozyme, and in order for the system to be self-sustaining, the circles would also have to encode the polymerase ribozyme itself (which is supplied separately in this paper and is not replicated). Nevertheless, this paper makes an important step, and continues to bring us closer to developing self-replicating RNA systems.Lines 122-126 – It is implied that triplets are better than monomers for rolling circle replication because triplets help to open up other double stranded regions. However, it is not obvious that this should be the case. To put a new triplet down you have to displace three bases from the displaced strand, whereas to put a monomer down you only have to displace one base. It is not easy to predict which of these is faster without measuring it. Furthermore, in the actual mechanism occurring here, there is no prior strand to be displaced at the point of attachment, because the displacement is occurring at the other side of the circle and it does not directly interfere with the attachment. So it is not clear whether this argument applies. Has replication of a circular strand actually been attempted with a monomer ribozyme? Is it known whether a triplet ribozyme is better than a monomer ribozyme on circular templates? If not, it would be better to avoid implying this.Figure 1 – the periodic effect seen in 1E is claimed to be due to the difference of accessibility of template bases on the inside and outside of the circle. However, the results are measured by averaging over many different circular templates. I would expect that different copies of a circular template would have different configurations and would not always have the same bases on the inside. So the inside-outside difference should average out. Could the variability of 1E be explained by variation in rates of addition according to the sequence of the template rather than an inside-outside effect? The sequence effect would be the same in multiple copies of the same template sequence. Is there a similar variability seen when copying linear templates?Figure 1C shows a TPR dimer. Is the polymerase actually in two parts? Is this important?Line 213 – It is not clear why the 9 bp primer goes straight to 18 bp. What happened to the lengths in between?Line 220 – The word "extended" is used to mean that the unhybridized portion is stretched. There is a possible confusion with extending a strand by ligation of a triplet. Maybe the use of a word like stretched is better?Line 226 – The simulations show that the double-stranded part of the circle is stiff and only covers roughly half of the circle. The point at which the primer extension occurs is therefore far away from the point at which the two strands separate. This is important for very short circles. For longer circles, the stiffness should be less relevant, and there will come a point where the whole circle becomes double stranded. There will then need to be a true strand displacement occurring very close to the point of primer extension. How long would we need the circle to be before it switches to a double-stranded circle? Does the stiffness effect seen here with the short circles make the primer extension reaction easier or more difficult than a true strand displacement reaction on a double stranded circle?Lines 228-33 – This paragraph is not very clear. The meaning is not coming through.Line 288 – "orbit" is an odd word. Is there a better one?Figure 4 – Overall Figure 4 is not clear.– I have not understood the notation n: 3E5, 2E5 etc.– For sequence A Pos 4, GAA is the darkest shade, so I am presuming the template is CUU (in the reverse direction). But the second darkest shade is GGC. Why should GGC bind to CUU more strongly than others (for example GGA)? I am not sure whether I have understood this diagram correctly.– Part C shows fold difference. It would be easier if rates where shown for linear and circular strands separately. Why is sequence D a worse template? Or maybe it is not worse – it's just that the ratio of circular to linear templates is lower? It is not easy to understand this.– Part D Figure 2 seems to show a double-stranded triplet being added. Why not just a single-stranded triplet.Figure 6D – It is unclear what is happening at each step. Particularly the backwards and forwards diagrams in step 4. Also, shouldn't the red strand be still attached to the blue circle before the cleavage occurs? The chemical structure in the middle is a bit distracting. I think the structure drawn in A is the same as D step 6. Maybe put parts A and D together and make B and C a separate figure?Line 436 – The reference to the virtual circular genome is misleading at this point. In the proposal of Zhou et al., there are no real circles, there are simply linear fragments that can be aligned to form a virtual circle. This does not fit with the rest of this paragraph. Either the reference to Zhou et al. should be omitted or it should be explained properly what the virtual circle proposal is.Reviewer #3:Technically the experiments are sound, really comprehensive, convincing and the paper is well written. The documentation  is very nice. The MD simulations nicely complement the experiments. Strong point is that the simulations address a qualitative question and are clearly directed to solve it. It is a preferable application of the MD technique. The basic methodology is correct, the standard AMBER OL3 force field appears appropriate, as the first choice multipurpose RNA version. It is known to lead to over-compacted unstructured ssRNA ensembles, as all biomolecular force fields that are good for folded biopolymers. For the double strand, the circle and their flexibility it should be an optimal choice. The simulations are quite short by contemporary standards, though I do not think their prolongation would change the essence of the findings.As noted above, I consider the experiments as very convincing. Strong point is that the accompanying simulations address a qualitative question and are clearly directed to solve it. It is a preferable application of the MD technique. So, I really like it, though I have some ideas for potential minor improvements, may be explanatory comments, all for supporting information.There are some occasional typos, e.g. l. 180 stand displacement, l. 182 this suggest. I think on l. 56. where progress in non-enzymatic synthesis is overviewed, the reference could be more balanced. Appears to me that some groups are represented by duplicate citations while some research is omitted, for example a recent progress in template-free non-enzymatic RNA polymerization of 3',5' cyclic nucleotides , https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/syst.202100017.The short "jumping" supplementary movies are difficult to follow, I assume it is because of the size of the movies. Would it be possible to create a few SI Figures showing details of the most interesting parts of the structure, to focus on the key details, to accompany the movie?2+ simulations up to 500 mM MgCl. First point, is this condition relevant to the origin of life? Second, inclusion of divalents into MD is always risky, as they sample poorly . 400 ns is not sufficient to converge Mg2+. In addition, divalents, especially the high charge density Mg2+, are beyond the pair-additive MM approximation. It is impossible to simultaneously balance ion hydration and inner vs. outer shell binding to different coordination sites with the simple MM models. Could the authors briefly comment on initial placement of the ions after equilibration and during MD? Was it always hexacoordinated outer-shell binding to the RNA? Could the authors in SI briefly comment on it, and also comment if they had some specific reasons to choose the Duboue-Dijon parameters over parameters that have been more commonly used in biomolecular simulations? As I am not sure these specific parameters were tested/calibrated for RNA interactions (but I am not fully familiar with the work). Again, as the results are qualitative, I do not expect any effect on basic outcome of the work, so I am not suggesting any new computations.In the simulations, lot of emphasis is paid on the Mg Essential revisions:Reviewer #1:In the origin of life scenario where RNA is assumed to be the first replicator, a key problem is how RNA can replicate itself. Or how can RNA polymerase copy itself, since copying requires an open flexible structure that can be read. While the polymerase needs to be a topological rigid structure in order to catalyse the RNA polymer.The manuscript describes how a special kind of synthesis of the RNA, rolling circle synthesis on small pieces of circular RNA, could template and build RNA strands. The method apparently could help in avoiding the strand inhibition problem where stable RNA duplex in long strands hinders replication.It is a bit unclear how, but I suspect this i down to the size of the ring, since the problem increase with the length of the polymer. The authors also touch on this themselves in their MD simulations, since the entropically unfavourable confinement of a string onto a circle can alleviate part of the problem. Nevertheless, the authors also show by MD that the rings become small tight structures that actually hinders replication.We welcome these comments. In the revised manuscript we have rewritten the relevant sections in order to clarify our arguments with respect to aspects of rolling circle synthesis that might aid RNA-catalyzed RNA replication.In brief, our argument relies on the conjecture (supported by our MD simulations) that in small RNA rings , increasing strain upon RNA synthesis can contribute to the dissociation of double-stranded (ds) RNA into single-stranded (ss) RNA at both the 5’- or the 3’-RNA ends. Only the 3’- (but not the 5’-) RNA end is extended by the triplet polymerase ribozyme (TPR) and this process is irreversible. Therefore, over time there will be an overall shift of the dsRNA segment around the circle in the 3’- direction resulting in a 5’-ssRNA “tail” of increasing length. The efficiency of this process relies critically on the speed and processivity of the triplet polymerase ribozyme (TPR) (which is currently poor) and its ability stabilize the RNA 3’-end bound to the circRNA template and extend it before it can dissociate again.Another potential issue is the fact that (as referee1 correctly observes), our MD simulations suggest RNA synthesis on scRNA templates stretches out the scRNA into an extended, rigid structure, which may be a poor template for replication. However, our experimental data suggest that rolling circle RNA synthesis is not only possible but can proceed over multiple full-length scRNA circles. We hypothesize that this reflects the fact that these rigid structures are likely in equilibrium with more relaxed structures, with more significant dissociation of the dsRNA segment from the circRNA template and / or kinks which relieve ring strain (even though these are not observed in our MD simulations) and it is those that can serve as templates for 3’-extension. Finally, although not supported by one-pot RCS experiments presented in Figure 4, it cannot be ruled out that the nascent strand comprises extension on two or even multiple scRNA templates. The precise mechanistic details do merit further study and this work is ongoing in our lab, but would lead too far for the current publication.The study depends on trinucleotide triphosphates (triplets) as substrates. They demonstrate a viroid like replication that show how a template (-) is able to make a mirror copy (+) of circular RNA by a rolling circle synthesis with out the need for enzymes or other catalyst apart from RNA itself.While the conclusion of the work becomes a bit muddled, but honest, the work is a very important piece that demonstrates the huge potential role of circular RNA in the very early stages of life.We thank the reviewer for his comments and have endeavored to make our conclusions as clear as possible. Furthermore, we have rewritten part of the Discussion section of the manuscript to increase clarity.I must confess I am very far off my field of competences. I have tried my best to understand the paper, and the methods involved, but obviously I cannot give much feedback on the methods used. So, I cannot suggest improvements on that part.For the most part, the paper is well written, and the structure is sound. I would strongly recommend publication.It was unclear to me how it was certain that it was actually rings. There is the MD simulations, but apart from the principle drawing they are obviously not circular in shape.Referee 1 raises an important point. Our study depends on the circularity of the RNA template molecules, i.e. small circular RNAs (scRNAs). We have therefore gone to some lengths to develop robust methods to efficiently generate such scRNAs, as well as to confirm their circularity experimentally. This is shown in Figure 1B and Supplementary Figure 1 . scRNAs are generated by ligation from linear RNAs and circularity is confirmed by exonuclease degradation (exoT). Only scRNA can resist degradation by exoT, as only circular RNAs do not present a free RNA 3’-end as a substrate . Circularity is further confirmed by electrophoretic mobility shift .To illustrate these points more clearly we have expanded the Figure legends for Figure 1B and Figure 1 —figure supplement 1, Figure 4 —figure supplement 1 and have also expanded the Materials and methods section describing experimental procedures to generate and purify scRNAs.There are some parts of the manuscript that too me is very difficult to understand. Some statements seem to reflect a community understanding that might be obvious for those working with similar system on a day to day basis, but for the general reader (where I put myself) this becomes gibberish. for instance:RCS has potentially unique properties with regards to the strand inhibition problem where RNA duplex melting in principle can be effected by continuous toehold strand displacement driven by nucleotide hybridization and the ratchet of nascent strand extension by triphosphate hydrolysis. In an idealized RCS mechanism, such strand invasion and displacement processes are both isoenergetic and coordinated to nascent strand extension , with rotation of the single-stranded RNA (ssRNA) preventing the build-up of topological tension . 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 circumventing the "strand inhibition problem" .It might not be possible to rephrase this so that everyone can understand. But I think the clarity of manuscript could be improved.We would like to apologize for failing to make our arguments sufficiently clear and free of jargon. We have now rephrased this passage in effort to make it both clearer as well as more accessible to the general reader. Please find below the rewritten section as found in the revised manuscript:“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 . 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.”But also because there is references to previous work where long statements does not make it more clear what is actually meant. for instance:Similar to what was described previously, RNA synthesis by the TPR best in the eutectic phase of water ice, due to beneficial reaction conditions for ribozyme catalysis such as reduced RNA hydrolysis and high ionic and RNA substrate concentrations . This was also the case on scRNA templates.Again, we would like to apologize for failing to make our arguments sufficiently clear to be easily understood. We have now rephrased this passage in an effort to make it both clearer as well as more accessible to the general reader. Please find below the rewritten section as found in the revised manuscript:2+ ions) present in the eutectic brine phase that arise by excluding solutes from growing ice crystals and remains liquid at subzero temperatures . Thus, all RCS experiments were carried out under eutectic conditions."“As described previously, RNA synthesis by the TPR is most efficient in the eutectic phase of water ice, due its beneficial reaction conditions for ribozyme catalysis . 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 , which describes the properties of the triplet polymerase ribozyme (TPR) and the general TPR performance on linear RNA templates, which form stable secondary structures (such as hairpins). The key observation is that the TPR can replicate through such RNA structures as a function of triplet concentration, while the standard mononucleotide RNA polymerase ribozyme (RPR) cannot.Referee 2 is of course correct in that triplets require the displacement of a 3 nt stretch from the primer / nascent strand 5’-end, whereas a mononucleotide RPR would require only a single base-pairing interaction to be disrupted. However, if this were the main energetic bottleneck of strand extension, surely the same argument would apply to template secondary structures, where again only one base-pairing interaction at the hairpin base would need to be disrupted compared to three for triplet invasion. However, our data show that only triplets endow the polymerase ribozyme with a general ability to invade and replicate through template secondary structures.While we do not know the precise mechanistic basis for this, the fact that secondary structure invasion is a cooperative effect that occurs as a function of triplet concentration suggests, that it may be based on the ability of triplets to bind to intermittently accessible template structures. Triplet binding would stabilize them in their open form enabling the ribozyme to covalently link them to the nascent strand. Indeed, extension is an irreversible process (a ratchet), while 5’-end displacement is an equilibrium process suggesting that given sufficient time even an inefficient extension “ratchet” will “win”.We of course accept that here may be other mechanisms for processive RNA synthesis on circular templates, such as e.g. tethering or topological linkage mechanism ). However, we note that Cojocaru and Unrau do not investigate beyond full length circle synthesis and it is therefore currently unclear if their ribozyme is merely more processive on an “open“ template or if its superior activity extends to strand displacement.We have now sought to explain our arguments more clearly in the revised manuscript .Has replication of a circular strand actually been attempted with a monomer ribozyme? Is it known whether a triplet ribozyme is better than a monomer ribozyme on circular templates? If not, it would be better to avoid implying this.We have not tested the mononucleotide RPR on circular RNA templates as – at the time this work was performed – all RPR variants required a hybridization tether to the template for processive RNA synthesis. On a circular RNA template this would likely cause both topological issues as well as – for full length synthesis – require displacement of the tether from the template for further extension. In contrast, the TPR does not require a tether for processive RNA synthesis by virtue of its non-catalytic t1 RNA subunit  and this – together with above (2.1) described properties of triplets with regards to template secondary structure invasion – were the reasons we sought to explore triplet-based RCS. Recently, a more advanced RPR version has been described that appears to use a σ factor-like initiation mechanism that enables processive synthesis specifically on much larger, circular RNA templates (ca. 200 nt), where strain may be a lesser concern . However, as discussed above (2.1), strand displacement was not investigated in this report.We have rewritten the relevant sections of the manuscript to clarify our argumentsFigure 1 – the periodic effect seen in 1E is claimed to be due to the difference of accessibility of template bases on the inside and outside of the circle. However, the results are measured by averaging over many different circular templates. I would expect that different copies of a circular template would have different configurations and would not always have the same bases on the inside. So the inside-outside difference should average out. Could the variability of 1E be explained by variation in rates of addition according to the sequence of the template rather than an inside-outside effect?This is a perceptive observation and we have considered the exact same problem. Our explanation, even though speculative, is that the primer binds first and remains bound stably and therefore orients the nascent strand in a particular configuration with decreasing flexibility as the double-stranded RNA segment grows (as indicated by the MD simulations). Indeed, with primer bound and stably oriented – as illustrated in Figure 1F, we would expect to see the observed banding pattern based on accessibility of the triplet junctions. Again, the positioning of the primer is supported by the MD simulations . As the template in this case is a UUC repeat, sequence variations (or variations in the rate of triplet addition according to the sequence of the template) are unlikely to account for the pattern observed. Indeed, we did not observe such a pattern in linear or non-repetitive circular template sequences. Furthermore, the same (or a very similar) periodic effect was observed on other repetitive circular templates, but not on their linear counterparts of identical sequence .The sequence effect would be the same in multiple copies of the same template sequence. Is there a similar variability seen when copying linear templates?When copying linear templates (of similar sequence as the circularized RNA template) the periodic effect is not seen.Figure 1C shows a TPR dimer. Is the polymerase actually in two parts? Is this important?Yes, the triplet polymerase ribozyme holoenzyme is a heterodimer comprising an active  RNA subunit (5TU) and an inactive subunit (t1). While the catalytic subunit by itself is a polymerase ribozyme, the non-catalytic subunit aids processive RNA synthesis . Therefore, the dimeric form of the TPR is the holoenzyme. This is important as the noncatalytic subunit is responsible for its ability to synthesize RNA in the absence of a template hybridization tether, which is specifically beneficial on circular RNA templates . An in-depth description of the TPR and is properties can be found in our previous paper . This has been specified in line 129-136 in the revised manuscript.Line 213 – It is not clear why the 9 bp primer goes straight to 18 bp. What happened to the lengths in between?In order to limit the number of MD runs, we concentrated on scRNAs with dsRNA segments reflecting later triplet additions  as we anticipated that ring strain would only begin to manifest itself in these molecules. Indeed, on scRNAs simulations with 9 bp and 18 bp of dsRNA, we observed an equally unbent double helical part, because the structure of the ssRNA part is longer and more relaxed and so it doesn’t exert any pulling force on the rest of the circle. We therefore did not model intermediate lengths 12 ( +1 triplet) and 15 (+ 2 triplets).Line 220 – The word "extended" is used to mean that the unhybridized portion is stretched. There is a possible confusion with extending a strand by ligation of a triplet. Maybe the use of a word like stretched is better?We thank the reviewer for this suggestion. “Extended” is indeed a questionable choice of word and liable to be confused with triplet extension. We have now replaced it with “stretched.”Line 226 – The simulations show that the double-stranded part of the circle is stiff and only covers roughly half of the circle. The point at which the primer extension occurs is therefore far away from the point at which the two strands separate. This is important for very short circles. For longer circles, the stiffness should be less relevant, and there will come a point where the whole circle becomes double stranded. There will then need to be a true strand displacement occurring very close to the point of primer extension. How long would we need the circle to be before it switches to a double-stranded circle?This is an important point. In the manuscript we suggest that this limit should be around the persistence length of RNA, which is about 200 bp. This mechanical property indicates the minimum length from which a polymer starts to behave flexibly, i.e. can significantly bend.Does the stiffness effect seen here with the short circles make the primer extension reaction easier or more difficult than a true strand displacement reaction on a double stranded circle?As discussed to some extent in (1.1) there are two conflicting mechanisms at work. On one hand, ring strain should aid strand displacement and therefore RNA synthesis beyond full length on a circular RNA template by destabilizing 5’-end hybridization and thereby aiding binding of triplets and extension. On the other hand, ring strain would be equally likely to destabilize 3’-end hybridization and thereby hinder extension and these two processes should therefore cancel each other out. Furthermore, there are other confounding factors such as restricted accessibility of some triplet junctions due to ring geometry , which may further reduce extension efficiency compared to a linear RNA template. But 3’-end extension by covalent triplet ligation is a unidirectional, irreversible process and therefore overall we observe primer extension, rolling circle synthesis and strand displacement.Lines 228-33 – This paragraph is not very clear. The meaning is not coming through.We would like to apologize for not making the parsing clear and thank Reviewer 2 for making us aware of this. We have now reprised the paragraph to the following in effort to clarify:“In the experimental data, we also observed an inhibitory effect for insertion of the final triplets  / 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’-end destabilization observed in the MD simulations , which would likely attenuate primer extension by the ribozyme. Note however that the extension efficiency recovered beyond full length , although at lower speed .”Line 288 – "orbit" is an odd word. Is there a better one?We have now replaced “orbit” throughout the manuscript with “full length circle synthesis”. We agree with referee 2 that orbit may be liable to misinterpretation.Figure 4 – Overall Figure 4 is not clear.– I have not understood the notation n: 3E5, 2E5 etc.– For sequence A Pos 4, GAA is the darkest shade, so I am presuming the template is CUU (in the reverse direction). But the second darkest shade is GGC. Why should GGC bind to CUU more strongly than others (for example GGA)? I am not sure whether I have understood this diagram correctly.The diagram represents the deep sequencing result of a one-pot extension reaction where all four templates were mixed, in the presence of the TRP and triplets . The reads from the four templates were assorted relative to position 3 (the first barcode triplet). For the rest of the positions, the diagram presents (in %) which triplets were identified. The notation “n:” stands for number of reads used for the analysis at each position. 3E5, 2E5 represent the number . This has now been specified in Figure 4B line 356-369 in revised manuscript.The darkness of the shading represents the amount (%) of triplets found at the noted position. As correctly stated in the comments, the (reverse order) template for pos 4 is CUU and indeed GAA is the expected correct triplet. The expected triplet for each position is marked with a box in the diagram. In pos 4, the expected GAA is darkest shade. However, as he sequencing data indicated, GGC clearly is also misincorporated at this position to some degree and this is represented in the figure. This has been specified in the text line 328-341 in revised manuscript.The overall fidelity of the TPR (per base) for linear templates is 97% , but positional fidelity can vary and GC-rich triplets can be particularly prone to misincorporation both in a templated context (due to e.g. G-U mispairing) as well as in a non-templated context, as triplet terminal transferase extension of free unpaired 3’- ends.– Part C shows fold difference. It would be easier if rates where shown for linear and circular strands separately. Why is sequence D a worse template? Or maybe it is not worse – it's just that the ratio of circular to linear templates is lower? It is not easy to understand this.The fold difference in fidelity that is shown in Part C, is calculated based on the fidelity (%) presented in B, thus the ratio of templates should not play a role. However, to ensure that the concentration and integrity (circular or linear) of the templates were validated (by gel purification and absorbance measurement) and an equal amount of each were added in the experiment.Calculation of plot in C is now specified in line 365-367 (in revised manuscript).– Part D Figure 2 seems to show a double-stranded triplet being added. Why not just a single-stranded triplet.A double stranded segment , or a template and a number of triplets) will stack and may form a substrate for non-templated terminal transferase addition by the TPR. We have not discussed this in the manuscript as more data would be needed for a conclusive mechanistic description. As this remains therefore a somewhat speculative model , we have now changed the figure to show a single stranded triplet as this is, as suggested, more intuitive. We have also added an explanation to the Figure 4 legend to the effect that the precise molecular nature of non-templated addition is currently unknown.Figure 6D – It is unclear what is happening at each step. Particularly the backwards and forwards diagrams in step 4. Also, shouldn't the red strand be still attached to the blue circle before the cleavage occurs? The chemical structure in the middle is a bit distracting. I think the structure drawn in A is the same as D step 6. Maybe put parts A and D together and make B and C a separate figure?We have changed the order in the figure so that the diagram with the reaction steps is presented before the data . In lines 424-430 (see revised manuscript) we have specified what the steps in the diagram denotes. Also, the Figure annotations for Figure 6A-D has been updated to fit the new Figure.Line 436 – The reference to the virtual circular genome is misleading at this point. In the proposal of Zhou et al., there are no real circles, there are simply linear fragments that can be aligned to form a virtual circle. This does not fit with the rest of this paragraph. Either the reference to Zhou et al. should be omitted or it should be explained properly what the virtual circle proposal is.We welcome this perceptive point and accept that our reference may be misleading in the context. However, while it is true that in the virtual circular genome proposal of Zhou et al., there are no real circles during the replication phase, Zhou et al. still propose circular RNAs arising from abiotic circularization reactions as an initial source of a circular genome. Nevertheless, we have now rewritten this section to clarify our arguments and removed the reference to Zhou et al.Reviewer #3:Technically the experiments are sound, really comprehensive, convincing and the paper is well written. The documentation  is very nice. The MD simulations nicely complement the experiments. Strong point is that the simulations address a qualitative question and are clearly directed to solve it. It is a preferable application of the MD technique. The basic methodology is correct, the standard AMBER OL3 force field appears appropriate, as the first choice multipurpose RNA version. It is known to lead to over-compacted unstructured ssRNA ensembles, as all biomolecular force fields that are good for folded biopolymers. For the double strand, the circle and their flexibility it should be an optimal choice. The simulations are quite short by contemporary standards, though I do not think their prolongation would change the essence of the findings.As noted above, I consider the experiments as very convincing. Strong point is that the accompanying simulations address a qualitative question and are clearly directed to solve it. It is a preferable application of the MD technique. So, I really like it, though I have some ideas for potential minor improvements, may be explanatory comments, all for supporting information.There are some occasional typos, e.g. l. 180 stand displacement.We have corrected this in the revised manuscript.l. 182 this suggest. I think on l. 56. where progress in non-enzymatic synthesis is overviewed, the reference could be more balanced. Appears to me that some groups are represented by duplicate citations while some research is omitted, for example a recent progress in template-free non-enzymatic RNA polymerization of 3',5' cyclic nucleotides , https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/syst.202100017.We have focused our introduction and citations mainly on enzymatic  polymerisation of RNA and believe we provide a fair and balanced account and citation of the main advances in that specific field. Where we have briefly alluded to prebiotic chemistry including references to non-enzymatic RNA replication, we have concentrated on chemical approaches that are closely analogous to the enzymatic chemistry e.g. polymerization via an (activated) 5’phosphate. But reviewer 3 is of course correct that we should not discount alternative prebiotic mechanisms as these may have similarly given rise to early (circular) RNA templates for enzymatic replication. We have now therefore expanded and modified the citations in the introduction to include e.g. the above-mentioned publication as well as others.The short "jumping" supplementary movies are difficult to follow, I assume it is because of the size of the movies. Would it be possible to create a few SI Figures showing details of the most interesting parts of the structure, to focus on the key details, to accompany the movie?We thank the reviewer for his suggestion. We have created Figure 3 —figure supplement 1 and 2 with a series of snapshots along the two trajectories showing the main transition events.2+ simulations up to 500 mM MgCl. First point, is this condition relevant to the origin of life?In the simulations, lot of emphasis is paid on the Mg2+ ions, RNA etc. are excluded from the growing water-ice crystals into a liquid brine (eutectic) phase that surrounds the ice crystals and remains liquid at subzero temperatures. This process is accompanied by a profound concentration and dehydration effect, which enhances RNA synthesis and reduces RNA degradation, but also increases counter-ion concentrations to the above levels (0.5M).As now outlined in more detail in the manuscript, the RNA polymerization activity of our triplet polymerase ribozyme is enhanced in the eutectic phase of water ice at subzero temperatures. Such eutectic phases from when water containing solutes freeze and solutes such as Mg2+ conditions with regards to the importance of eutectic ice phases in the experimental section.The formation and presence of water ice on the early earth can of course not be proven, but is not implausible under currently favored geochemical scenarios for example at the poles or as part of seasonal or diurnal temperature variations. We have outlined our thinking on the beneficial properties of such ice phases in detail in one of the cited refs  and now also discuss it in more detail within the main body of the revised manuscript. Furthermore, we now include an explicit reference to the rationale for simulations at high Mg2+. In addition, divalents, especially the high charge density Mg2+, are beyond the pair-additive MM approximation. It is impossible to simultaneously balance ion hydration and inner vs. outer shell binding to different coordination sites with the simple MM models. Could the authors briefly comment on initial placement of the ions after equilibration and during MD? Was it always hexacoordinated outer-shell binding to the RNA? Could the authors in SI briefly comment on it, and also comment if they had some specific reasons to choose the Duboue-Dijon parameters over parameters that have been more commonly used in biomolecular simulations? As I am not sure these specific parameters were tested/calibrated for RNA interactions (but I am not fully familiar with the work). Again, as the results are qualitative, I do not expect any effect on basic outcome of the work, so I am not suggesting any new computations.Second, inclusion of divalents into MD is always risky, as they sample poorly . 400 ns is not sufficient to converge Mg2+ represents a challenge for empirical force-field calculations due to strong polarization and charge-transfer effects. Indeed, nonpolarizable Mg2+ parameters tend to overestimate the formation of contact ion pairs by a factor of 2 to 3 . We chose the parameters developed by Jungwirth and coworkers  because they drastically reduce ion−ion interactions with respect to ion−water interactions . They account for the quantum effects by assuming a mean field approach using the so-called Electronic Continuum Correction (ECC), which is numerically implemented by rescaling the charge of multivalent ions, in the case of Mg2+ to 1.5.We thank the reviewer for raising all these important points. We agree that MgWe agree with the reviewer that 400 ns is short for an accurate description of the ion environment. However, we have added Figure 3 —figure supplement 6 showing reasonable convergence of ion location around RNA within the first and second hydration shell for the time course of our simulations.We have added this extra information into the revised manuscript .


The high mutation rate leads to high genetic variability and fast evolution, providing an adaptive potential regarding new cellular receptors and hosts [Life, several researchers have studied or reviewed this group of RNA viruses with a specific focus on their ecology, evolution, and epidemiology.RNA viral genomes are generally small genomes that rarely exceed 10 kb in size. Moreover, these genomes are characterized by high mutation rates  RNA viruses was the acquisition of ExoN exoribonuclease (Nsp14). The RNA 3′ end mismatch excision activity of this enzyme promotes replication fidelity, and genome expansion in CoVs is believed to be at least partially mediated by Nsp14 gene acquisition . MutatioIn this context, phylogenetic analysis provides evidence that the evolution of CoVs includes several events of interspecies transmission linked to recombination events. For instance, all three novel zoonotic human CoVs  have been exposed to recombination events in their evolutionary history ,10,11. TPrior host jumps of coronaviruses to humans, bats and birds have been identified as natural animal reservoirs for coronaviruses, while civets and other mammals such as camels have been identified as intermediate amplifying hosts. For example, the emergence of Middle-East Respiratory Syndrome Coronavirus (MERS-CoV) into humans involved viral exchange between bats and camels before transmission to humans was possible ,15,16. ISelection occurring on emerging variants of coronaviruses strongly contributes to the adaptation that occurs following a shift from one host to another. The high levels of genetic variation combined with the large population sizes of coronaviruses provide an ideal genetic landscape for selection to optimize the host colonization. Selection on mutations affecting the spike protein seems to play the most important role in adaptation process since spike protein is involved in the host cellular entry and the evasion of host cellular immune responses. However, there is evidence that selection on mutations affecting other encoded proteins also contributes to innate immune escape. In this issue, Mandilara and colleagues highlighted the important role of Nsp15 and Nsp16 RNA in modifying enzymes during the innate immune escape of coronaviruses .As with other viruses, CoVs also depend on cellular host factors and pathways for successful replication. Valuable information from interactome studies led to the rapid discovery of host RNA binding proteins that bind to viral RNA, regulating its replication and translation . Since SDespite the genetic diversity among different coronavirus clades and the infection of different organisms and cell types, specific host signaling pathways that drastically affect their propagation are manipulated in a common way. For instance, as reviewed by Gioti and colleagues in this issue, animal coronaviruses employ similar molecular mechanisms in order to hijack the originally protective apoptotic signaling of the host and stimulate apoptosis at later stages of infection in order to favor viral dissemination by cell breakdown .During the COVID-19 pandemic, mutations of SARS-CoV-2 that are associated with high transmissibility have been subjected to positive selection . SeveralNot surprisingly, given the unfolding pandemic caused by SARS-CoV-2, this Special Issue also attracted more clinically oriented research. The significant morbidity and mortality of COVID-19, as well as the huge burden it posed to clinical services globally, led to an unprecedented global research mobilization that resulted, within just a few months, in the production of research outputs and knowledge that normally requires years to be accumulated. This intense research production came with some limitations. For example, numerous clinical trials evaluated the same treatments, unwarrantedly exposing numerous patients to experimental treatments that were later proved to be ineffective or associated with adverse events . Moreover, other clinical trials omitted endpoints that are important to patients . In thisIn summary, this Special Issue represents a collection of articles that study and discuss different aspects of the ecology, evolution, and epidemiology of coronaviruses, as well as evolving research on clinical aspects on COVID-19. We hope that the diverse articles of this issue cover a significant part of coronavirus research in these fields. A better understanding of the molecular mechanisms that govern genetic evolution and adaptation will help us to develop a better epidemiological surveillance and battle the coronavirus-driven threats to public health more efficiently. It is obvious that more remains to be discovered about the manipulation of cellular signaling pathways by the viral proteins. The limitations of our knowledge on the immune escape mechanisms of coronaviruses pose challenges for the next years in terms of growing a more comprehensive understanding of these adaptation processes and developing new therapeutic approaches.


Development of distant metastasis is the main cause of deaths in prostate cancer (PCa) patients. Understanding the mechanism of PCa metastasis is of utmost importance to improve its prognosis. The role of exosomal long noncoding RNA (lncRNA) has been reported not yet fully understood in the metastasis of PCa. Here, we discovered an exosomal lncRNA HOXD-AS1 is upregulated in castration resistant prostate cancer (CRPC) cell line derived exosomes and serum exosomes from metastatic PCa patients, which correlated with its tissue expression. Further investigation confirmed exosomal HOXD-AS1 promotes prostate cancer cell metastasis in vitro and in vivo by inducing metastasis associated phenotype. Mechanistically exosomal HOXD-AS1 was internalized directly by PCa cells, acting as competing endogenous RNA (ceRNA) to modulate the miR-361-5p/FOXM1 axis, therefore promoting PCa metastasis. In addition, we found that serum exosomal HOXD-AS1 was upregulated in metastatic PCa patients, especially those with high volume disease. And it is correlated closely with Gleason Score, distant and nodal metastasis, Prostatic specific antigen (PSA) recurrence free survival, and progression free survival (PFS). This sheds a new insight into the regulation of PCa distant metastasis by exosomal HOXD-AS1 mediated miR-361-5p/FOXM1 axis, and provided a promising liquid biopsy biomarker to guide the detection and treatment of metastatic PCa. Prostate cancer (PCa) is the second commonly diagnosed malignancy and one of the leading cause of male cancer-related death worldwide . MetastaExosomes are membranous microvesicles ranging 40–150 nm in dimension, which are found in various human fluids including, but not limited to, blood, urine, and bile . RecentlPreviously we constructed two castration resistant prostate cancer (CRPC) cell models, LNCaP-AI and LNCaP-Bic, by exposing LNCaP cells to continuous androgen deprived medium or antiandrogen drug. These two cell lines displayed similar biological and molecular characteristics with clinical CRPC . We demon = 5). Surprisingly, we found that the serum exosomes from metastatic patients significantly enhanced the motility of PCa cells, as evaluated by transwell and wound healing assays . Collectively, these findings indicate that exosomal HOXD-AS1 may participated in the metastasis of PCa.To investigate the effect of serum exosomes from PCa patients on PCa cells, we treated LNCaP and PC-3 cells with exosomes from localized and metastatic patients , as analyzed by Starbase database  detection has been applied clinically as the marker to predict the sensitivity of novel anti-androgen regimens [Exosomal RNAs are emerging as novel diagnostic bio-makers for its non-invasiveness and stable in body fluids . Exosomaregimens . Moreoveregimens –43. Hereregimens –46. As aIn summary, our findings revealed evidence of the mechanism in which CRPC cell-derived exosomal HOXD-AS1 promoted PCa metastasis by modulating miR-361-5p/FOXM1 axis. We also reported that serum exosomal HOXD-AS1 detection could be applied as a marker for metastatic disease, as well as predicting the prognosis of PCa patients. Our study not only identifies a crucial mechanism of exosomal lncRNA-mediated intercellular communication from CRPC cells to the TME, which endowed common PCa cell with metastatic features, but also develops a potential non-invasive diagnostic approach for PCa.The cell lines used in this study were the human prostate cancer cells LNCaP and PC-3 , and the CRPC cell line LNCaP-Bic and LNCaP-AI as previously reported , 47. Andst Affiliated Hospital of Kunming Medical University. All the samples were pathologically diagnosed as prostate adenocarcinoma by two pathologists. The clinical features of the patients are listed in Table st Affiliated Hospital of Kunming Medical University. Informed consent was obtained from all participants prior to sample collection.A total of 36 and 9 cases paraffin embedded PCa and benign prostate hypertrophy (BPH) tissues were obtained by surgery or needle biopsy, and 130 cases of serum samples were collected from treatment-naïve patients after their initial pathological diagnosis from the 1-∆∆Ct method. The transcription level of GAPDH was used as an internal control for mRNAs, and U6 was used as control for miR-361-5p. All specific method and primers are listed in Supplementary The experiments was conducted as previously described . The relThe pCDNA3.1-HOXD-AS1 overexpressing vector was constructed, and stable knockdown of HOXD-AS1 in PCa cells were obtained from our previous report . The humn = 10, estimated 40–50% proportion of metastasis from preliminary experiment.). BALB/c-nu mice  were anesthetized and randomly divided into 4 groups, the pre-treated cells were slowly inoculated into the left cardiac ventricle at 5 × 105 cells in 100 μL of PBS per mice. Then either PBS or 20 μg indicated exosomes at 50 μl volume were injected intra-cardiac under anesthesia weekly at the same time bioluminescence imaging was conducted. Osteolytic lesions were identified on radiographs as radiolucent lesions in the bone. Each bone metastasis was scored as previously reported [All mouse experiments were approved by the Institution of Animal Care and Use Committee of Kunming Medical University  and housed as previously reported . For thereported , 52 basereported .RNA in situ hybridization, isolation of exosomes, transmission electron microscopy (TEM), nanoparticle tracking analysis, exosomes tracking, Western Blot and antibody information, in vitro assays, and other methods are described in Supplementary Author Contribution FormFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8Figure S9Table S1Table S2Table S3Table S4Supplementary Materials and MethodsSupplementary Figure LegendsSupplementary ReferenceschecklistAuthor Contribution statement