Anna Marie Pyle Reviewing Editor; Yale University, United States In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Ribozyme-catalysed RNA synthesis using triplet building blocks" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Nebojsa Janjic (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The most important thing to improve is the written clarity of the manuscript. There was a lot of agreement between the reviewers' comments so we attach their full reviews below for your attention. All their concerns should be attended to.

Reviewer #1:

Attwater et al. report an advance in efforts to develop a robust self-replicating RNA by presenting an example of an RNA polymerase ribozyme (RPR) capable of transcribing RNA from structured RNA templates of substantial length with fairly good fidelity. This RPR uses a trinucleotide building block (in contrast to NTPs used by standard polymerases), that the authors speculate could have implications in the evolution of mRNA decoding by the ribosome.

The major findings from this work are as follows:

1) The authors exploit the ligase activity in the catalytic core of their previously reported RPR Z and evolve a suite of catalytic sequences capable of extending an RNA primer using RNA triplets as substrates. Through further iterations of in-vitro selection for triplet dependent RPRs, an RNA sequence emerged without any catalytic activity itself, but it enhanced triplet extension by these newly evolved RPR sequences (named t2 through t6) through a bimolecular interaction between the catalytically proficient sequence. Further rounds of selection using the cofactor t1 resulted in the most efficient triplet RPR, t5+1 which was used for downstream studies. This RPR could use structured hairpins as templates, even those with Tm >90oC that were intractable to previous RPRs that use NTPs as substrates. The authors demonstrate the efficiency of this RPR by transcribing both + and – strands of the Broccoli aptamer, a structured and fluorescent RNA.

2) The authors used t5+1 RPR to replicate fragments of itself (both + and – strands), using triplet building blocks (and in some cases hexamers). The complete active RPR could be reconstituted from the five individual components of the RPR transcribed by the same RPR. Although this replicase ribozyme is still not able to replicate itself in its entirely, it does give rise to a functioning replicase by using building blocks as small as trimers, Previous efforts had primarily used the ligase activity of the RPRs to assemble oligonucleotides representing their sequence to assemble a 'replica' of itself. Considering the challenges of self-replication, even incremental advances in the right direction are significant.

Overall this work introduces a new RPR system that brings the field closer to a self-replicating system, which is one of the cornerstones of Origin of life research. The authors explore properties of this replicase including fidelity and catalytic efficiency and explain the molecular basis for these properties. This work also introduces trinucleotides as plausible substrates on which ancient RNA-based replicases could have acted. Considering the relative ease of non-enzymatic polymerization of monomers into triplets, the presence of these triplets in a prebiotic setting seems plausible. The current work is detailed and the authors have explored multiple facets of the new system they have setup. This new RPR presents a major advance in terms of its ability to create a fully functional copy of itself (albeit in fragments). As such, this would be a great addition to the current understanding of RNA-based self-replication systems.

1) Overall, the manuscript was very difficult to follow. It is up to the authors to make their presentation as simple and direct as possible. Given the availability of supporting information, there is no reason why the authors could not add schemes where appropriate to clarify their presentation, logic, and workflow.

2) Regarding the dimerization, it is not clear how the authors implicated the 5'-hairpin to begin with, leading to the experiment that showing that a single mutation in that loop inhibits dimerization.

3) The assertion that the two RNAs form a 1:1 complex is not quantitatively substantiated from Figure 3B.

4) Considering their success in replicating structured hairpins and achieving primer free synthesis of an 18 nt strand (β+), it would be interesting (though not necessary for publication) to see how much of the Broccoli RNA could be transcribed without the addition of a primer sequence. If this is not possible, the authors could demonstrate the complete replication of a functional RNA of tractable length, like the Hammerhead ribozyme, to illustrate the ability of their trinucleotide RPR to replicate entire sequences of functional RNAs by exclusively using triplets as substrates.

5) The fidelity analysis is based on testing 12 of the possible 64 trinucleotides, so one cannot be convinced of the generality of their observations and inferences. The authors need to underscore this in the discussion of fidelity. The authors show that increased concentrations of triplets help fidelity, however in the absence of the 'correct' triplet, it is likely that RPR will incorporate 'mismatch' triplets. The authors should comment on this aspect.

Reviewer #2:

1) In this paper, the authors demonstrate interesting and novel results that provide further evidence for RNA's capacity to act as prebiotic genetic material. The key feature associated with this study involved using trinucleotide triphosphates (triplets) rather than NTPs as substrates for a novel RNA polymerase ribozyme. The use of triplets allowed structured templates to be unfolded and in some cases avoided the need for primers, circumventing an ongoing obstacle to RNA self-replication. This study thus makes some significant steps towards identifying a truly self-replicating prebiotic biopolymer. Literature precedents are well-discussed, and the results are interpreted in a detailed and convincing manner. I would nonetheless encourage the authors to try to make these important results as accessible as possible to readers who are unfamiliar with in vitro evolution.

Specific comments:

2) Subsection “in vitro evolution of triplet polymerase activity”, last paragraph. The use of "in-ice evolution" (a technique developed by the authors) is reported. A sentence could be added describing why this technique is being used / why it is necessary.

3) For certain experiments (e.g. subsection “Fidelity of triplet-based RNA synthesis”), random triplet pools were used. What kind of triplet pools were used in other sequence-copying experiments? Were they non-random? This needs to be more clearly articulated.

4) "… we found that using modified substrates with a disrupted minor groove hydrogen bond acceptor at the 3rd position…" – What is the modification? This should be stated in the body-text.

5) What is the scope of substrates for the t5+1 ribozyme? Triplets reportedly incur a lower entropic cost than NTPs (Discussion, third paragraph), but what would happen when a mixture of the two were used? Would both the NTPs and triplets be incorporated?

If these points are satisfactorily addressed, then I am in favour of acceptance.

Reviewer #3:

Attwater et al. used an in vitro evolution method to enhance the trace amount of ligase activity with short (3-nucleotide) substrates inherent in the truncated version of the Z RPR ribozyme (called "Zcore"). This was achieved by adding a random region of 30 nucleotides at the 3' end of Zcore and a template at the 5' end, followed by selection for variants able to extend a primer hybridized to the 5' template with two 5'-triphosphate triplets and ultimately ligate the extended primer to the 5' terminal triphosphate. After 7 rounds of activity enrichment, the authors identified a sequence that represented 25% of the activity-enriched pool called "type 0" that, upon truncation to its minimal active sequence ("0core"), was able to catalyze multiple cycles of templated ligation with triplet substrates, including the ability to extend thought double-stranded regions of the template by cooperative invasion through the secondary structure of the template (this was not achievable with monomer substrates, NTPs).

After additional 14 rounds selection, the composition of the pool showed considerable changes: type 0 sequence had disappeared and was replaced by six new types of sequences, types 1-7. Remarkably, the most prevalent type 1 sequence (50% of the pool) was completely inactive and types 2-6 were less active than the activity enriched pool. This mystery was solved by the observation that a truncated version of the type 1 sequence, while being inactive, can form augment the activity of type 2-6 ribozymes by forming heterodimeric complexes. In the presence of type 1 sequence, the best stand-alone ribozyme, type 5, no longer required the template to be tethered to the 5' end of the type 5 ribosome, in a remarkable example of an intermolecular complex between the dimeric type 1:type5 ribozyme, the untethered primer template, and the triplet substrates. Further optimization of the type 5 ribozyme by re-randomization of the previously fixed 3' end sequences and reselection in the presence of minimal type 1 sequence (called "1") resulted in the final heterodimeric holoenzyme called "t5+1. t5+1 was able to catalyze the extension through templates with Tm values of 93 ⁰C, and was capable of generating both the functionally active Broccoli RNA aptamer and its complementary template. Finally, t5+1 was able to replicate its own t5 sequence split into five segments, mostly from triplets, with some help from longer substrates (hexamers) needed for two of the segments, thus falling short of having the capacity for a full replication cycle, but nonetheless, exhibiting impressive catalytic efficiency.

The surprising observation that t5+1 can extend primers in both the canonical 5'-3' as well as the reverse 3'-5' direction raises the potential for primer extension from short sequences in a primer-free manner, therefore potentially solving the "primer problem" (depletion of longer but presumably sparse primers needed for replication in prebiotic conditions). Impressive overall positional fidelity of more than 97% was achieved by counter-selection for mispairing triplets with a clever inclusion of excess 3'-deoxy terminator triplets during late rounds of selection. Attwater et al. mapped the region responsible for fidelity to the 3' domain of t5. Further reduction in mis-incorporation comes from cognate triplet-triplet interactions have a self-correcting feature of countering the effect of triplets with strong base-pairing tendency.

The authors make a strong case that triplets represent relatively short and yet powerful substrates able to invade highly stable structural features in RNA templates. The evolution of a heterodimeric ribozyme capable of generating functional RNAs with triplet substrates represents a major accomplishment that advances the plausibility of RNA-catalyzed RNA replication in prebiotic environment. Despite its considerable length, as well as its remarkable breadth and scope, the manuscript reads like a riveting novel. It is a tour de force scientific accomplishment, and as such, it will be of considerable interested to a wide audience of readers of eLife. [Editors’ note: this reviewer’s minor comments have not been included.]