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.

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.

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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.

Viroids are transcriptional parasites composed entirely of a circular RNA genome and are considered the simplest infectious pathogens known in nature. They lack protein-coding regions in their genome and can be completely replicated in ribosome-free conditions (Daròs et al., 1994; Diener, 2003; Fadda et al., 2003; Flores et al., 2009). They can comprise a circular RNA genome of as little as ~300 nt in, for example, Avsunviroidae encoding a Hammerhead ribozyme responsible for maturation by resecting the replicated RNA genome (Flores et al., 2000). The resected viroid genome is then ligated (circularized) by a host protein (e.g., tRNA ligase) (Nohales et al., 2012). Due to the simplicity of this replication strategy, viroids have been suggested to represent possible ‘relics’ from a primordial RNA-based biology (Diener, 1989; Flores et al., 2014). Indeed, circular RNA genomes would present a number of potential advantages for prebiotic RNA replication, including increased stability by end protection (Litke and Jaffrey, 2019) and a reduced requirement for specific primer oligonucleotides to sustain replication (Attwater et al., 2018; Szostak, 2012). A circular RNA genome also resolves the end replication problem, that is, the potential loss of genetic information from incomplete replication in linear genomes. Circular RNA structures can self-assemble from RNA mononucleotides through wet-dry cycling (Hassenkam et al., 2020) providing potential initiation points for primordial RNA replication. Thus, viroid-like replication systems are plausible candidates to have emerged as the simplest genetic systems.

Here, we have explored to what extent such a potentially prebiotic replication strategy can be carried out by RNA alone. Our data show that RNA can indeed perform RCS on scRNA templates yielding concatemeric RNA products, which can be processed (i.e., resected) 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 (e.g., a second µHHz) encoded in the (−)-strand akin to the mechanism used by natural viroids (Flores et al., 2000).

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 (i.e., strand displacement). 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 (Mutschler et al., 2015).

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 (Wawrzyniak et al., 2017) and in biotechnology (Daubendiek et al., 1995; Givskov et al., 2016; Kristoffersen, 2017; Mohsen and Kool, 2016). dsDNA persistence length is somewhat shorter than dsRNA (dsDNA: 45–50 nm [140–50 nt] vs. dsRNA 60 nm [200 nt]) and stacking interactions in dsDNA are weaker than in dsRNA (Kebbekus et al., 1995; Svozil et al., 2010). Therefore, dsDNA may more readily adopt a circular shape or allow internal kinks to alleviate build-up of strain or to adopt strong bends (Wolters and Wittig, 1989). Nevertheless, we would expect a similar strand displacement effect would play a part in RCS on small circular DNA templates. Indeed, in both cases, RCS proceeds efficiently for circular genomes ranging from a few hundred nt to over 1.5 kb (Mohsen and Kool, 2016). In contrast, RNA-catalyzed polymerization (current maximum approx. 200 nt products; Attwater et al., 2013) is currently limited to RCS on scRNAs. A more efficient RNA-catalyzed RCS-based replication strategy will likely require improvements to the ribozyme polymerase catalytic activity, speed, and processivity as well as the design of the template. Improvements to ribozyme polymerase processivity, which is known to be poor (Johnston et al., 2001; Lawrence and Bartel, 2003), might have the greatest impact and might be realized either through tethering or other topological linkages to the circular template (Cojocaru and Unrau, 2021). A more processive polymerase ribozyme should also result in less non-templated triplet terminal transferase activity, which appears to be a consequence of slow RCS extension and is likely aggravated by dissociation of the 3′-end. Thus, more efficient RCS may also require the stabilization of the 3′-end triplet junction in the ribozyme active site in the same way as primer/nascent strand termini are stabilized within the active sites of proteinaceous RNA- and DNA polymerase (Chim et al., 2018; Houlihan et al., 2020). Finally, introduction of secondary structure motifs in the RNA nascent strand might drive increased 5′-end dissociation (e.g., through formation of stable hairpin structures) relieving strain at the 3′-end.

Larger 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 (He et al., 2017; Tupper and Higgs, 2021). In this context, we reasoned that RCS might provide favourable properties: synthesis and strand displacement on a circular template can in principle proceed essentially iso-energetically as base-pairing (H-bonding/stacking) interactions broken at the 5′-end during strand displacement are continuously compensated for by new base-pairing interactions formed at the nascent strand 3′-end. In turn, this enables an open-ended formation of template-coupled stochiometric excess of single-stranded RNA product strand to encode functions to further aid replication as we show here with resection and recircularization by an encoded ribozyme.

In 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 (Figure 3). However, the MD simulations also suggest that strain is non-directional destabilizing both nascent strand 5′- as well as 3′-ends equally. Thus, the potential advantages of scRNA RCS seem to be tempered by opposing effects such as strain as well as reduced template accessibility due to circular RNA ring geometry (Figure 1). Nevertheless, we find that a viroid-like replication strategy can be accomplished by RNA catalysis alone, with one ribozyme performing RCS on circular RNA templates yielding concatemeric RNA products, which can be processed (i.e., resected) and recircularized by a second ribozyme. Future improvements in polymerase ribozyme activity and processivity may allow all necessary components of such a replication cycle to be encoded on a circular RNA ‘genome’ and propagated by self-replication and -processing reactions.

All data generated or analyzed in this manuscript is supplied within the manuscript or supporting file; Source Data files containing original unedited gels images as well as numeric data have been provided for Figures 1,2,4 and 5, as well as figure supplements when relevant. Modelling data and sequencing data are provided as described in the data availability section in the manuscript.

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Decision letter after peer review:

Thank you for submitting your article "Rolling Circle RNA Synthesis Catalysed by RNA" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Timothy Nilsen as Reviewing Editor and James Manley as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Jiri Sponer (Reviewer #3).

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 (Blanco et al., 1989; Daubendiek et al., 1995), with rotation of the single-stranded RNA (ssRNA) preventing 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 circumventing the "strand inhibition problem" (Tupper and Higgs, 2021).

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 (Attwater et al., 2010). 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 (the composed Figures etc.) 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?

In the simulations, lot of emphasis is paid on the Mg²⁺ 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 (which is further exacerbated by the lack of bulk background due to the small periodic box, which may lead to glassy-like ion behavior around the solute). 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.

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 (small circular RNAs, scRNAs), 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 (and described in Supplementary information Materials and Methods). 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 (Figure 1B). Circularity is further confirmed by electrophoretic mobility shift (Figure 1 —figure supplement 1 and Figure 4 —figure supplement 1).

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 (Blanco et al., 1989; Daubendiek et al., 1995), with rotation of the single-stranded RNA (ssRNA) preventing 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 circumventing the "strand inhibition problem" (Tupper and Higgs, 2021).

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 (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.”

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 (Attwater et al., 2010). 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:

“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 (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 arise by excluding solutes from growing ice crystals and remains liquid at subzero temperatures (Attwater et al., 2010). Thus, all RCS experiments were carried out under eutectic conditions."

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:

[…]

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.

Referee 2 raises an important point and we welcome the opportunity to clarify our arguments.

The implication that triplets are able to “open up” double-stranded RNA regions is based on data in our previous publication (Attwater et al., 2018), 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 (as explored e.g. by (Cojocaru and Unrau, 2021)). 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 (see also comments above).

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 (Attwater et al., 2018) 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 (Cojocaru and Unrau, 2021). 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 arguments

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?

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 (Figure 3A). 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 (see Figure 2 —figure supplement 1B).

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 (catalytic) 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 (see Attwater et al., 2018). 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 (as already outlined in more detail in 2.2). An in-depth description of the TPR and is properties can be found in our previous paper (Attwater et al. 2018). 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 (+3 triplets, 18 / + 4 triplets, 21 etc.) 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 (see Figure 1), 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 (+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’-end 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).”

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 (CUG, AUA, CCA, CCC, AAA, CAC, GGG, UUA, UCC, GGC, AUC, GAU, CGC and GAA). 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 (3*10^5, 2*10^5 etc.). 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% (Atwater et al. 2018), 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 (consisting of e.g. two complementary triplets (triplet and anti-triplet, e.g. GGC and CCG), 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 (although consistent with the data obtained), 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 (see new Figure 6). 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 (the composed Figures etc.) 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 (RNA-catalyzed) 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.

In the simulations, lot of emphasis is paid on the Mg²⁺ simulations up to 500 mM MgCl. First point, is this condition relevant to the origin of life?

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⁺ 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).

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 (Attwater et al., 2010) 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⁺ conditions with regards to the importance of eutectic ice phases in the experimental section.

Second, inclusion of divalents into MD is always risky, as they sample poorly (which is further exacerbated by the lack of bulk background due to the small periodic box, which may lead to glassy-like ion behavior around the solute). 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.

We thank the reviewer for raising all these important points. We agree that 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 (Fingerhut et al., 2021). We chose the parameters developed by Jungwirth and coworkers (Duboue-Dijon et al., 2018) because they drastically reduce ion−ion interactions with respect to ion−water interactions (see new Figure 3—figure supplement 6; Fingerhut et al; Kirby and Jungwirth, 2019). 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 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 (Method section and Figure 3 – Supplement Figure 6).

Author details

1. Emil Laust Kristoffersen

   MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom

   Present address

   Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark

   Contribution

   Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8965-8201

2. Matthew Burman

   Department of Physics, University of York, York, United Kingdom

   Contribution

   analysed data, discussed results and co-wrote the manuscript

   Competing interests

   No competing interests declared

3. Agnes Noy

   Department of Physics, University of York, York, United Kingdom

   Contribution

   analysed data, discussed results and co-wrote the manuscript, performed dynamics simulation, analysed data, discussed results and co-wrote the manuscript

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0673-8949

4. Philipp Holliger

   MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom

   Contribution

   analysed data, discussed results and co-wrote the manuscript, analysed data, discussed results and co-wrote the manuscript

   For correspondence

   ph1@mrc-lmb.cam.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3440-9854

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