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Cited by 279Origins and Evolution of LifeRNA and protein synthesis mechanismsEvolution and Genetic Dynamics

In-ice evolution of RNA polymerase ribozyme activity

Open access
Attwater, James, Wochner, Aniela, Holliger, Philipp·2013·Nature Chemistry
DOI 10.1038/nchem.1781PMC PMC3920166
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Abstract

Mechanisms of molecular self-replication have the potential to shed light upon the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial ‘RNA World’. However, existing polymerase ribozymes lack the capacity to synthesise RNAs approaching their own size. Here we report the in vitro evolution of such catalysts directly in the RNA-stabilising medium of water-ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesise RNA in ices at temperatures as low as −19°C. Combination of cold-adaptive mutations with a previously described 5′ extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication.

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Crossref · 10.1038/nchem.1781

Cites

34 of 34 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 36 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    After two days of incubation, Y made more RNA in ice at −7°C than at 17°C.

    “Indeed, Y synthesised more RNA in ice (at −7°C) than at 17°C, after just two days’ incubation.”
    Quote in the paperSubstantial claim
  • Findingcitation

    During one week in ice, Y extended more than 35% of primers by 12 nucleotides before pausing at a difficult template block.

    “During incubation for one week in ice, Y extends over 35% of primers by 12 nts, before pausing at a challenging template sequence block16.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Attwater, J. et al. Chemical fidelity of an RNA polymerase ribozyme. Chem. Sci. 4, 2804–2814 (2013). source

      Not checked in the free run.

  • Findingown data

    The ribozyme could carry out substantial RNA synthesis at temperatures as low as −19°C.

    “Substantial RNA synthesis by the ribozyme could be observed down to temperatures as low as −19°C (Fig. 2b).”
    Quote in the paperSubstantial claim
  • Findingown data

    The C60U mutation from the ambient-temperature-evolved Z ribozyme did not improve activity in ice more than at ambient temperature.

    “As expected, the C60U mutation, derived from the Z ribozyme evolved at ambient temperatures, did not improve activity in ice any more than at ambient temperatures.”
    Quote in the paperSubstantial claim
  • Findingown data

    Combining cold-adaptive mutations with a previously described 5′ extension produced a polymerase ribozyme that can accurately synthesise an RNA longer than itself, up to 206 nucleotides.

    “Combination of cold-adaptive mutations with a previously described 5′ extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication.”
    • Also stated in: § Introduction · plain
      “and derived a ribozyme - tC9Y - able to accurately synthesise RNA oligomers longer than the ribozyme itself using a favourable RNA template sequence at 17°C.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source

      Not checked in the free run.

  • May combine two claims. 32
  • Classified as probably not a substantial claim. 3

  • May combine two claims.

    Findingown data

    Reverting individual Y mutations reduced polymerase activity, and comparisons of extension at ambient temperature and in ice identified G93A and C97A as the main cold-specific contributors, with U72G contributing only slightly.

    “Reversion greatly reduced polymerase activity, but its impact differed at different temperatures: ratios of quantified extensions at ambient temperatures and in ice indicate that G93A and C97A are the key mutations responsible for boosting cold-specific activity, with U72G providing a minor contribution (Fig. 3b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Y outperformed R18 on several templates and showed substantial polymerase activity in ice at −7°C.

    “Y outperformed the parent R18 ribozyme on a range of templates (Supplementary Fig. S3d), but in particular displayed substantial polymerase activity in ice at −7°C (Fig. 1c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Y exceeded R18 by about fourfold at 17°C, by more than 15-fold at −7°C in ice, and by more than 30-fold in supercooled solution.

    “While Y outperformed R18 by fourfold at 17°C (as judged by gel densitometry of average extension per primer15,22), at −7°C the differential rose to more than 15-fold in ice, and more than 30-fold in a supercooled solution.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [15] Attwater, J., Wochner, A., Pinheiro, V. B., Coulson, A. & Holliger, P. Ice as a protocellular medium for RNA replication. Nature Commun. 1, 76 (2010). source

      Not checked in the free run.

    • cites [22] Muller, U. F. & Bartel, D. P. Improved polymerase ribozyme efficiency on hydrophobic assemblies. RNA 14, 552–562 (2008). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    C8 differed from R18 by only three mutations, U72G, G93A and C97A, and these together gave superior in-ice polymerase activity.

    “C8 exhibited only three mutations with respect to the parent R18 sequence (U72G, G93A, C97A, Supplementary Fig. S3a), which together provided superior in-ice polymerase activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Screening by the ribozyme polymerase plate assay found two clones, C30 and C8, with improved in-ice primer extension, and both outperformed R18 on two primer/template combinations.

    “RPA screening identified two polymerase ribozyme clones with improved in-ice primer extension activity, C30 and C8, both of which outperformed R18 on two different primer/template combinations (Supplementary Fig. S1c, d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Adding the previously described C60U mutation from Z to C8 gave a moderate further increase in activity and produced ribozyme Y.

    “Introducing the previously described C60U mutation11 from Z gave a moderate further boost to activity (Supplementary Fig. S3b, c) yielding the ribozyme ‘Y’ (Fig. 1b).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Engineering C30 produced ribozyme W, which resembled Z in phenotype but carried an additional set of moderately cold-adaptive mutations.

    “Engineering C30 yielded the ribozyme ‘W’ which displayed a phenotype related to that of ‘Z’ (an R18 ribozyme variant with increased sequence generality evolved independently at 17°C11), but which possessed an additional set of moderately cold-adaptive mutations (Supplementary Fig. S2).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Across temperatures, Y had a lower temperature optimum than R18 and retained significant activity at −7°C in ice and in supercooled solutions.

    “Comparison of RNA synthesis by R18 and Y over a range of temperatures revealed a lower temperature optimum for Y that supported significant activity at −7°C in ice and in supercooled solutions (Fig. 2a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The selected sequences shared features including low secondary-structure propensity, reduced G content, and bias against -DD- dinucleotides except -UU-.

    “These (likely interdependent) characteristics include a low tendency for secondary structure formation, a reduced G-content as well as bias against -DD- (D= A, G, U) dinucleotides (with the exception of -UU-) (Fig. 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Five ~50-nucleotide templates were chosen from the selected pool, and four of them, A50, B50, D50 and E50, could be extended to full length both at ambient temperature and in ice.

    “From this pool (Supplementary Table S3) we randomly selected five ~50 nt template sequences (A50, B50, C50, D50 and E50, Fig. 5a), of which four (A50, B50, D50, E50) could be extended to full length (~+50 nts) both at ambient temperatures (Fig. 5b) and in ice (Supplementary Fig. S8b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    R18 prefers the shorter template I-s, on which Y performs worse than on the longer template I.

    “Furthermore, the parental R18 ribozyme shows a preference for a shorter template (I-s) on which Y performs worse than on the longer template I”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In ice, the template pool stayed diverse over four selection rounds, and templates able to direct full-length synthesis emerged.

    “in ice the template sequence pool remained diverse over four rounds of selection, during which templates capable of directing full-length syntheses emerged (Supplementary Fig. S8a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sanger sequencing of full-length products showed that the Y mutations gave a modest fidelity improvement, to 98.3% at 17°C versus 97.3% for tC19, and to 94.8% in ice versus 93.4% for R18.

    “Sanger sequencing of full length products11 (Supplementary Table S2) indicated that the Y mutations conferred a modest fidelity improvement at 17°C to 98.3% compared to 97.3% for tC1911, and in ice to 94.8% compared to 93.4% for R1815.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source

      Not checked in the free run.

    • cites [15] Attwater, J., Wochner, A., Pinheiro, V. B., Coulson, A. & Holliger, P. Ice as a protocellular medium for RNA replication. Nature Commun. 1, 76 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    On long templates made of repeated template I, R18 achieved only limited polymerisation, whereas Y synthesised extension products up to +30 nucleotides in ice.

    “Whereas R18 could only achieve limited polymerisation upon such templates, Y was able to synthesise long extension products up to +30 nt in ice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Y performs better when a long downstream single-stranded template is present, without needing specific base-pairing to a template motif.

    “Indeed, Y performs better when a long downstream single-stranded template is available, without requiring specific base-pairing with a template sequence motif (Fig. 3c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    On favourable templates, tC9Y has a termination probability per incorporation of 0.012-0.025, with about 97.5% of primers extended past each successive nucleotide after a week.

    “With ~97.5% of primers extended beyond each successive nucleotide by the end of the week-long incubation (Supplementary Fig. S5a), tC9Y exhibits a termination probability per incorporation on these favourable templates of 0.012-0.025.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Because of the Y mutations, tC9Y is the fastest and most efficient polymerase ribozyme reported so far, making 63-nucleotide products in 16 hours and 206-nucleotide products in 60 hours.

    “Due to the influence of the Y mutations, tC9Y is also the fastest and most efficient polymerase ribozyme described so far, yielding 63 nt products after only 16 hours and 206 nt products after 60 hours (Supplementary Fig. S4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The newly selected in-ice ribozymes showed both cold-specific adaptations, enabling synthesis in ice down to −19°C, and generic improvements independent of temperature and medium.

    “The novel in-ice selected RNA polymerase ribozymes (Supplementary Fig. S9) displayed both cold-specific adaptations, allowing ribozyme-catalysed RNA synthesis to proceed in ices at temperatures as low as −19°C (Fig. 2), and generic i.e. temperature- and medium-independent activity improvements”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In-ice selection yielded a diverse set of novel long (50-nucleotide) RNA sequences that were efficient templates for enzymatic RNA synthesis both in ice and at ambient temperature.

    “To better understand this, we isolated a diverse repertoire of novel long (50 nt) RNA sequences by in-ice selection that proved efficient templates for enzymatic RNA synthesis both in ice and at ambient temperatures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    This termination probability is about twofold better than tC19 and fourfold better than tC19Z, previously the most efficient polymerase ribozymes.

    “This is an approximately 2-fold improvement on tC19 (0.019-0.05) and 4-fold improvement on tC19Z (0.03-0.1) (Supplementary Fig. S6), previously the most efficient polymerase ribozymes described11.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Combining the three in-ice-selected mutations with the C60U mutation and the 5′ ssC19 tag produced tC9Y, which synthesised RNA polymers up to 206 nucleotides, longer than its own 202 nucleotides.

    “Combining these three mutations from the in-ice ribozyme selection with the previously identified C60U mutation and the 5′ terminal ssC19 tag11 yielded a novel polymerase ribozyme tC9Y that could synthesise RNA polymers of up to 206 nts in length (Fig. 4a), longer than itself (202 nts), illustrating the synthetic potential of polymerase ribozymes.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In ice, tC9Y long-range synthesis was much slower and initiation of frozen extension in this setup was inefficient.

    “As well as proceeding much more slowly (Supplementary Fig. S4a), initiation of frozen extension in this setup was inefficient.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Illumina analysis of 1.17×10^5 positions gave 97.7% fidelity for full-length products and 97.4% overall fidelity when incomplete products were included.

    “Analysis of 1.17×105 positions revealed a 97.7% fidelity for full length products, and a slightly lower overall fidelity of 97.4% when including all incomplete extension products (Supplementary Fig. S7a, Table S2).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ice is one of several heterogeneous prebiotic media that could compartmentalise RNA, which is a prerequisite for evolution.

    “Ice is also one of a range of heterogeneous prebiotic media (including aerosols, lipidic membranes and porous rock17-20) with the potential to effect RNA compartmentalisation, a prerequisite for evolution6.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [17] Dobson, C. M., Ellison, G. B., Tuck, A. F. & Vaida, V. Atmospheric aerosols as prebiotic chemical reactors. Proc. Natl Acad. Sci. USA 97, 11864–11868 (2000). source

      Not checked in the free run.

    • cites [18] Chen, I. A., Salehi-Ashtiani, K. & Szostak, J. W. RNA catalysis in model protocell vesicles. J. Am. Chem. Soc. 127, 13213–13219 (2005). source

      Not checked in the free run.

    • cites [19] Budin, I. & Szostak, J. W. Expanding roles for diverse physical phenomena during the origin of life. Annu. Rev. Biophys. 39, 245–263 (2010). source

      Not checked in the free run.

    • cites [20] Monnard, P. A. Catalysis in abiotic structured media: an approach to selective synthesis of biopolymers. Cell. Mol. Life Sci. 62, 520–534 (2005). source

      Not checked in the free run.

    • cites [6] Szostak, J. W., Bartel, D. P. & Luisi, P. L. Synthesizing life. Nature 409, 387–390 (2001). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    R18 is not capable of self-replication because its synthetic capacity is limited, partly by weak primer/template duplex binding, and because it is unstable in the high Mg2+ concentration needed for polymerase activity.

    “However, the activity of R18 falls short of that required for self-replication in both its limited synthetic capacity (exacerbated by a low affinity for substrate primer/template duplex14) as well as its poor stability in the presence of the high concentration of Mg2+ ions (200 mM) required for polymerase activity9,15,16.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [14] Lawrence, M. S. & Bartel, D. P. Processivity of ribozyme-catalysed RNA polymerization. Biochemistry 42, 8748–8755 (2003). source

      Not checked in the free run.

    • cites [9] Johnston, W. K., Unrau, P. J., Lawrence, M. S., Glasner, M. E. & Bartel, D. P. RNA-catalysed RNA polymerization: accurate and general RNA-templated primer extension. Science 292, 1319–1325 (2001). source

      Not checked in the free run.

    • cites [15] Attwater, J., Wochner, A., Pinheiro, V. B., Coulson, A. & Holliger, P. Ice as a protocellular medium for RNA replication. Nature Commun. 1, 76 (2010). source

      Not checked in the free run.

    • cites [16] Attwater, J. et al. Chemical fidelity of an RNA polymerase ribozyme. Chem. Sci. 4, 2804–2814 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The activity improvements include both generic and ice-specific components, and the ice-specific component comes mainly from cold adaptation rather than from ice surfaces or structural features.

    “These results indicate both generic as well as ice-specific improvements in RNA polymerase ribozyme activity with the latter arising primarily from an adaptation to cold temperatures rather than to ice surfaces or structural features.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    R18 is an approximately 200-nucleotide ribozyme derived from a de novo ligase ribozyme and can extend RNA primers by up to 14 nucleotides using NTPs.

    “R18 is a ribozyme, approximately 200 nucleotides (nt) long, derived from a ligase ribozyme generated de novo from a random RNA sequence pool12,13, and can catalyse the templated extension of RNA primers by up to 14 nucleotides (nts) using nucleoside triphosphates (NTPs)9.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [12] Bartel, D. P. & Szostak, J. W. Isolation of new ribozymes from a large pool of random sequences. Science 261, 1411–1418 (1993). source

      Not checked in the free run.

    • cites [13] Ekland, E. H., Szostak, J. W. & Bartel, D. P. Structurally complex and highly active RNA ligases derived from random RNA sequences. Science 269, 364–370 (1995). source

      Not checked in the free run.

    • cites [9] Johnston, W. K., Unrau, P. J., Lawrence, M. S., Glasner, M. E. & Bartel, D. P. RNA-catalysed RNA polymerization: accurate and general RNA-templated primer extension. Science 292, 1319–1325 (2001). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Ice can enhance polymerase ribozyme activity and stability even though R18 was evolved at 22°C and is poorly adapted to the low temperature of the ice phase.

    “We had previously shown that ice, a biphasic medium, could enhance both polymerase ribozyme activity and stability15 - despite the poor adaptation of the R18 polymerase ribozyme (evolved at 22°C) to the low temperatures of the ice phase (−7°C).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [15] Attwater, J., Wochner, A., Pinheiro, V. B., Coulson, A. & Holliger, P. Ice as a protocellular medium for RNA replication. Nature Commun. 1, 76 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductreasoning

    The ice-CBT protocol used a diluted extension buffer whose concentration by eutectic phase formation restores an optimal eutectic composition and reduces bead aggregation.

    “The concentration effect of eutectic phase formation restores an optimal eutectic phase composition from this diluted extension buffer, reducing bead aggregation within the eutectic phase.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Ribozymes stayed ligated to beads during extension, which improved local primer extension.

    “Ribozymes remained ligated to beads during extension, improving local primer extension.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    The in-ice CBT selection used three initial low-stringency rounds, then recombination and five additional rounds of increasing stringency to explore sequence space and remove detrimental mutations.

    “To effectively explore sequence space using our modestly sized mutant repertoire (5×107) we exploited the strategies of neutral drift and recombination using three initial rounds of in-ice CBT at low stringency (requiring primer extension by 3-5 nucleotides for RCA signal, to deplete the sequence pool of detrimental mutations), followed by recombination and five additional rounds of in-ice CBT”
    Quote in the paperSubstantial claim
    • Rests on an assumption: A modest random mutant library combined with neutral drift and recombination can effectively explore sequence space.

  • May combine two claims.

    Interpretationcitation

    The crystal structure of the Class I RNA ligase suggests that this linker sequence is positioned to interact with the downstream RNA template and could strengthen the ribozyme's sequence-general contacts with the upstream duplex, enhancing primer/template binding.

    “Examination of the crystal structure of the Class I RNA ligase29,30, from which the catalytic core of the ribozyme polymerase is derived, suggests this sequence is well-positioned to interact with the downstream RNA template (Fig. 3a), and could augment the sequence-general contacts the ribozyme makes with the upstream duplex16,29,31 to enhance primer/template binding.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [29] Shechner, D. M. et al. Crystal structure of the catalytic core of an RNA-polymerase ribozyme. Science 326, 1271–1275 (2009). source

      Not checked in the free run.

    • cites [30] Shechner, D. M. & Bartel, D. P. The structural basis of RNA-catalysed RNA polymerization. Nature Struct. Mol. Biol. 18, 1036–1042 (2011). source

      Not checked in the free run.

    • cites [16] Attwater, J. et al. Chemical fidelity of an RNA polymerase ribozyme. Chem. Sci. 4, 2804–2814 (2013). source

      Not checked in the free run.

    • cites [31] Muller, U. F. & Bartel, D. P. Substrate 2′-hydroxyl groups required for ribozyme-catalysed polymerization. Chem. Biol. 10, 799–806 (2003). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    The ssC19 sequence extension enhances long RNA synthesis by helping the ribozyme tether to the 5′ end of the template.

    “the 5′-‘ssC19’ sequence extension (5′-GUCAUUG-), previously shown to enhance synthesis of long RNAs by facilitating ribozyme tethering to the 5′ end of the template11.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    This study demonstrates in-ice directed evolution of RNA function and adaptation of RNA polymerase ribozyme activity to the eutectic phase of water ice.

    “In this study we have demonstrated the in-ice directed evolution of RNA function, showing adaptation of RNA polymerase ribozyme activity to the conditions of the eutectic phase of water ice (Fig. 1).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: When an aqueous solution containing ions and RNA freezes, the solutes are excluded from growing ice crystals and concentrated into an interstitial eutectic brine.
34 of 34

  • The paper relies on this without support.

    When an aqueous solution containing ions and RNA freezes, the solutes are excluded from growing ice crystals and concentrated into an interstitial eutectic brine.

    “Ice formation from an aqueous solution comprising solutes such as ions and RNA results in a non-uniform, biphasic system whereby the solutes, including RNA, are excluded from the growing ice crystals and are concentrated in an interstitial brine, called the eutectic phase.”

  • The paper relies on this without support.

    A modest random mutant library combined with neutral drift and recombination can effectively explore sequence space.

    “To effectively explore sequence space using our modestly sized mutant repertoire (5×107) we exploited the strategies of neutral drift and recombination”

  • The paper relies on this without support.

    The 95 selected template sequences are representative enough to reveal general features that affect RNA template replicability.

    “Analysis of 95 selected template sequences revealed a consistent set of attributes that appear to critically influence the replicability of a given RNA sequence.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • When the reaction is performed in the eutectic phase of water ice, even longer polymers can be obtained because of increased concentration of reactants and reduced rate of RNA degradation under near-frozen conditions.

    “When the reaction is performed in the eutectic phase of water ice, even longer polymers can be obtained because of the increased concentration of reactants and reduced rate of RNA degradation under these near-frozen conditions”

    From Protocells and RNA Self-Replication (2018) · cites this paper as Attwater et al. 2013

    The Results section, “Synthesis of long RNAs in ice and at ambient temperatures” (page not available), reports products up to 118 nucleotides in ice, versus 206 nucleotides at 17°C, and says frozen synthesis was slower and less efficient. Although the paper says the eutectic phase concentrates solutes and ice can enhance ribozyme stability, its measured product lengths contradict the claim that this reaction yielded longer polymers in ice. (LLM, conf=0.88)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • Recently synthesized RNA-dependent RNA polymerase (replicase) ribozymes are highly complex and quite long, longer than the maximal length allowed by Eigen's paradox.

    “However, recently synthesized RNA-dependent RNA polymerase (replicase) ribozymes [105,106,107] are highly complex and quite long (longer than the maximal length allowed by Eigen’s paradox).”

    From Ecology and Evolution in the RNA World Dynamics and Stability of Prebiotic Replicator Systems (2017) · cites this paper as [106]

    The Discussion (page not provided) describes a 202-nucleotide polymerase ribozyme with a catalytic core and a processivity domain, supporting the description of a long, structurally complex ribozyme. It does not state that this length exceeds a maximum imposed by Eigen’s paradox. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Prior forms of the class I polymerase generally had difficulty extending past a misincorporated nucleotide, so full-length products contain fewer mutations compared with partial-length products.

    “Prior forms of the class I polymerase generally had difficulty extending past a misincorporated nucleotide, so the full-length products contain fewer mutations compared with the partial-length products (10, 13).”

    From An RNA polymerase ribozyme that synthesizes its own ancestor (2020) · cites this paper as [13]

  • Polymerase ribozymes selected in vitro over decades now have sufficiently high fidelity to accurately copy sequences 80-200 nucleotides long.

    “Polymerase ribozymes have been selected in vitro over decades, showing improvement over a series of selections, and now have sufficiently high fidelity to accurately copy sequences 80–200 nucleotides long [94–100].”

    From The RNA world as a model system to study the origin of life (2015) · cites this paper as [100]

  • Icy cold conditions enhance synthetic RNA polymerase ribozyme activity and shield the macromolecule from hydrolysis.

    “In the laboratory, cold conditions in icy environments promote the activity of synthetic RNA polymerase ribozymes, while also protecting the macromolecule from hydrolysis.”

    From Environmental Adaptation from the Origin of Life to the Last Universal Common Ancestor (2018) · cites this paper as Attwater et al. 2013

Cites

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2 of the 36 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. Atkins, J. F., Gesteland, R. F. & Cech, T. R. (eds) RNA Worlds (Cold Spring Harbor Laboratory, 2011).

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Authors
Attwater, J., Wochner, A., & Holliger, P.
Affiliations
MRC Laboratory of Molecular Biology (GB)
Corresponding author
Holliger, Philipp
ORCID
Venue
Nature Chemistry
Publisher
Springer Science and Business Media LLC
Published
2013-10-20
Volume
5
Issue
12
Pages
1011-1018
ISSN
  • 1755-4330 (ISSN-L)
  • 1755-4349
DOI
10.1038/nchem.1781
PMC
PMC3920166
OpenAlex
W2075152880
Semantic Scholar
5ce5403ee8b97dff394e91c0cecbffb2129a3e2d
Cited by
279
References
36
Citations by year
2026: 14 · 2025: 23 · 2024: 19 · 2023: 20 · 2022: 17 · 2021: 29 · 2020: 11 · 2019: 21 · 2018: 25 · 2017: 29 · 2016: 27 · 2015: 22 · 2014: 20 · 2013: 1
FWCI
12.58
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Chemistry (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Topics
  • Physical Sciences › Physics and Astronomy › Astronomy and Astrophysics › Origins and Evolution of Life
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Evolution and Genetic Dynamics
Keywords
RNA self-replication, RNA world, origin of life, cold adaptation, subzero temperatures, RNA synthesis, in vitro evolution, water ice
MeSH terms
Adaptation, Biological, Base Sequence, Cold Temperature, Ice, Models, Genetic, Molecular Sequence Data, Nucleic Acid Conformation, RNA, RNA-Dependent RNA Polymerase, RNA, Catalytic, Evolution, Molecular

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-04.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-04.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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