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In-ice evolution of RNA polymerase ribozyme activity
Open accessAbstract
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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“Indeed, Y synthesised more RNA in ice (at −7°C) than at 17°C, after just two days’ incubation.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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“During incubation for one week in ice, Y extends over 35% of primers by 12 nts, before pausing at a challenging template sequence block16.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
What the sources say
- Not checked yetcites [16] Attwater, J. et al. Chemical fidelity of an RNA polymerase ribozyme. Chem. Sci. 4, 2804–2814 (2013). source
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“Substantial RNA synthesis by the ribozyme could be observed down to temperatures as low as −19°C (Fig. 2b).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Abstract
What the sources say
- Not checked yetcites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source
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- Also stated in: § Introduction · plain
In the paper
“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.”
- Also stated in: § Introduction · plain
“Harnessing the compartmentalised bead-tagging (CBT) RNA selection strategy11 directly in ice, we isolated RNA polymerase ribozymes adapted to frozen conditions”
§ In-ice evolution of RNA polymerase ribozyme activity > Abstract
What the sources say
- Not checked yetcites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source
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- 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.
- Also stated in: § Introduction · plain
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“Thirdly, we found a striking deviation of the dinucleotide frequencies from those expected (correcting for the biased nucleotide composition) (Fig. 5d).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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“tC9Y copied these templates with an overall fidelity comparable to the I-n repeat template (Supplementary Fig. S8c, Table S2).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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“The G93A and C97A mutations that provide the main cold-specific activity boost are located in the single-stranded linker region of the ribozyme between the catalytic core (1-90) and the processivity domain (105-187).”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
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“Firstly, the majority of the template sequences exhibited a low propensity to form stable secondary structure elements (as judged by mfold26).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
What the sources say
- Not checked yetcites [26] Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003). source
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“In ice (−7°C), long-range synthesis by tC9Y only yielded products up to 118 nucleotides long (Supplementary Fig. S5b).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
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“However, the majority (>85%) of incomplete extension products terminated in the correct residues”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
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“Analysis of 95 selected template sequences revealed a consistent set of attributes that appear to critically influence the replicability of a given RNA sequence.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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In the paper
“Indeed, extension ‘blocks’ are frequently observed preceding template stretches lacking C are frequently observed16.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
What the sources say
- Not checked yetcites [16] Attwater, J. et al. Chemical fidelity of an RNA polymerase ribozyme. Chem. Sci. 4, 2804–2814 (2013). source
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“Conversely, RNA polymerisation often proceeds efficiently on template regions with regularly spaced C residues (such as template I-n).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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“Furthermore, termination patterns indicated that some primers were extended in an ssC19-independent manner (Supplementary Fig. S5c).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
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“It is notable that although these template selections harnessed the ssC19 tag, the templates’ beneficial properties extended to both the Y as well as the parental R18 RNA polymerase ribozyme (Supplementary Fig. S8d).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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“Secondly, we observed a clear and progressive trend of the template nucleotide composition away from parity towards a reduced G and an increased C content (Fig. 5c).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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“This new engineered tC9Y polymerase ribozyme proved exceptionally good at synthesising very long RNAs on these favourable templates, yielding RNA products up to 206 nucleotides long (Fig. 4a, b) at 17°C.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
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“All dinucleotides lacking C (except -UU-) were underrepresented”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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“Y performed even more proficient primer extension on a template (II) with a different downstream sequence (Fig. 3c), continuing up to a couple of bases from its 5′ end.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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In the paper
“However, existing polymerase ribozymes lack the capacity to synthesise RNAs approaching their own size.”
§ In-ice evolution of RNA polymerase ribozyme activity > Abstract
What the sources say
- Not checked yetcites [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 yetcites [10] Zaher, H. S. & Unrau, P. J. Selection of an improved RNA polymerase ribozyme with superior extension and fidelity. RNA 13, 1017–1026 (2007). source
- Not checked yetcites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source
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In the paper
“This is unlikely to be mediated by sequence-specific hybridisation, as Y performed even more proficient primer extension on a template (II) with a different downstream sequence (Fig. 3c), continuing up to a couple of bases from its 5′ end.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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“As CTP is efficiently incorporated opposite template G9, this trend does not reflect the elimination of inefficient template bases but rather the tendency of G-rich templates to promote the formation of highly stable secondary structures including G-rich hairpin motifs (through both G-C and G·U pairing) and potentially G-quadruplexes.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
What the sources say
- Not checked yetcites [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
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In the paper
“the strong sequence dependence of RNA synthesis in general remains an obstacle to self-replication.”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
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“suggesting that non-processive synthesis as well as degradation of the ribozyme and single-stranded template in the Mg2+-rich buffer were likely responsible for the majority of the observed terminations.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
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In the paper
“suggesting that Y may be able to productively interact with downstream single-stranded template RNA to achieve enhanced polymerisation in ice.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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In the paper
“G93A and C97A may be involved in low-temperature interactions with the RNA template, improving primer/template duplex binding.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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In the paper
“Indeed, the total 2.5% termination rate per position (Supplementary Fig. S5a) suggests just a ~1 / 300 chance of misincorporation-induced termination per position during these tC9Y-catalysed RNA syntheses.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
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“highlighting the potential of water-ice (and perhaps other heterogeneous media) as a favourable environment for the emergence of ribozyme phenotypes that may not be accessible under ambient conditions.”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
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“This may be due to altered dynamics and a reduced functionality of the ssC19 tethering function - optimized for ambient conditions - under the altered temperature and salt conditions in ice, greatly reducing overall synthetic efficiency.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
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“The need to copy both its complementary minus and plus strands during a replication cycle points to conflicting pressures with regards to nucleotide composition and secondary/tertiary structure formation needed for ribozyme activity.”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
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“Such compositional limitations are reminiscent of those observed in nonenzymatic contexts32, highlighting the analogous constraints upon both processes arising from the distinct stacking and hydrogen bonding properties of the natural nucleobases.”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
What the sources say
- Not checked yetcites [32] Joyce, G. F. & Orgel, L. E. Non-enzymatic template-directed synthesis on RNA random copolymers. Poly(C,A) templates. J. Mol. Biol. 202, 677–681 (1988). source
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“as the abundant C residues appeared to function to space out A, U and G, suggesting an important role for intermittent interactions to unpaired template C for productive template binding and/or translocation.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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“In early RNA replication, adaptive pressures would have acted not only on the replicase ribozyme itself, but on the replication system as a whole, including the replication templates.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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- It may state two separate claims as one. 32
- Probably not a substantial claim. 3
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
What the sources say
- Not checked yetcites [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 yetcites [22] Muller, U. F. & Bartel, D. P. Improved polymerase ribozyme efficiency on hydrophobic assemblies. RNA 14, 552–562 (2008). source
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
What the sources say
- Not checked yetcites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
What the sources say
- Not checked yetcites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source
plaindirectSingle case- Asserted: passes — The authors put it forward as true.
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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In the paper
“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”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of RNA polymerase ribozyme templates
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
What the sources say
- Not checked yetcites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source
- Not checked yetcites [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
hedgeddirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Whereas R18 could only achieve limited polymerisation upon such templates, Y was able to synthesise long extension products up to +30 nt in ice.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“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”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
What the sources say
- Not checked yetcites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
What the sources say
- Not checked yetcites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“As well as proceeding much more slowly (Supplementary Fig. S4a), initiation of frozen extension in this setup was inefficient.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Introduction
What the sources say
- Not checked yetcites [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 yetcites [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 yetcites [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 yetcites [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 yetcites [6] Szostak, J. W., Bartel, D. P. & Luisi, P. L. Synthesizing life. Nature 409, 387–390 (2001). source
hedgedconditional- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. The paper’s conclusions may not depend on it.
None of the sources cited for this claim have been checked yet.
In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Introduction
What the sources say
- Not checked yetcites [14] Lawrence, M. S. & Bartel, D. P. Processivity of ribozyme-catalysed RNA polymerization. Biochemistry 42, 8748–8755 (2003). source
- Not checked yetcites [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 yetcites [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 yetcites [16] Attwater, J. et al. Chemical fidelity of an RNA polymerase ribozyme. Chem. Sci. 4, 2804–2814 (2013). source
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Generic and ice-specific adaptations
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Introduction
What the sources say
- Not checked yetcites [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 yetcites [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 yetcites [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
plain- Asserted: passes — The authors put it forward as true.
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In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Introduction
What the sources say
- Not checked yetcites [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
hedgeddirect- Asserted: passes — The authors put it forward as true.
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Materials and methods > In ice ribozyme selection
plaindirect- Asserted: passes — The authors put it forward as true.
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In the paper
“Ribozymes remained ligated to beads during extension, improving local primer extension.”
§ In-ice evolution of RNA polymerase ribozyme activity > Materials and methods > In ice ribozyme selection
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim. It may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“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”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > In-ice selection of ribozyme polymerase activity
plaindirect- Asserted: passes — The authors put it forward as true.
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- Rests on an assumption: A modest random mutant library combined with neutral drift and recombination can effectively explore sequence space.
None of the sources cited for this claim have been checked yet.
In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
What the sources say
- Not checked yetcites [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 yetcites [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 yetcites [16] Attwater, J. et al. Chemical fidelity of an RNA polymerase ribozyme. Chem. Sci. 4, 2804–2814 (2013). source
- Not checked yetcites [31] Muller, U. F. & Bartel, D. P. Substrate 2′-hydroxyl groups required for ribozyme-catalysed polymerization. Chem. Biol. 10, 799–806 (2003). source
hedgedconditional- Asserted: passes — The authors put it forward as true.
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In the paper
“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.”
§ In-ice evolution of RNA polymerase ribozyme activity > Results > Synthesis of long RNAs in ice and at ambient temperatures
What the sources say
- Not checked yetcites [11] Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalysed transcription of an active ribozyme. Science 332, 209–212 (2011). source
plaindirect- Asserted: passes — The authors put it forward as true.
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Probably not a substantial claim.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“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).”
§ In-ice evolution of RNA polymerase ribozyme activity > Discussion
boosteddirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
- 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.
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.”
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?
The cited source doesn’t support the claim as written.
In the citing paper
“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
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“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]
Experimental — accuracy not yet measuredIn the citing paper
“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]
In the citing paper
“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]
In the citing paper
“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
In the citing paper
“While colder temperatures would slow down RNA polymerization rates, the enhanced stabilization and reduced degradation of the ribozyme counteract this effect, permitting synthesis of remarkably long molecules (Attwater et al. 2013;Attwater et al. 2010).”
From Environmental Adaptation from the Origin of Life to the Last Universal Common Ancestor (2018) · cites this paper as Attwater et al. 2013
Citations
Cites
Mfold web server for nucleic acid folding and hybridization prediction
Zuker, M. (2003) · Nucleic Acids Research · doi:10.1093/nar/gkg595 · Cited by 13,404 · cited 1×
“The G93A and C97A mutations that provide the main cold-specific activity boost are located in the single-stranded linker region of the ribozyme between the catalytic core (1-90) and the processivity domain (105-187).”
BackgroundGilbert, W. (1986) · Nature · doi:10.1038/319618a0 · Cited by 2,721 · cited 1×
“The G93A and C97A mutations that provide the main cold-specific activity boost are located in the single-stranded linker region of the ribozyme between the catalytic core (1-90) and the processivity domain (105-187).”
BackgroundGilbert, W. (1986) · Nature · Cited by 1,941
Szostak, J. W., Bartel, D. P., & Luisi, P. L. (2001) · Nature · doi:10.1038/35053176 · Cited by 1,681
Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions.
Powner, M. W., Gerland, B., & Sutherland, J. D. (2009) · Nature · doi:10.1038/nature08013 · Cited by 1,383 · cited 1×
“The G93A and C97A mutations that provide the main cold-specific activity boost are located in the single-stranded linker region of the ribozyme between the catalytic core (1-90) and the processivity domain (105-187).”
BackgroundIsolation of New Ribozymes from a Large Pool of Random Sequences
Bartel, D. P., & Szostak, J. W. (1993) · Science · doi:10.1126/science.7690155 · Cited by 900 · cited 1×
“The G93A and C97A mutations that provide the main cold-specific activity boost are located in the single-stranded linker region of the ribozyme between the catalytic core (1-90) and the processivity domain (105-187).”
BackgroundRNA-catalyzed RNA polymerization: Accurate and general RNA-templated primer extension.
Johnston, W. K., Unrau, P. J., Lawrence, M. S., Glasner, M. E., & Bartel, D. P. (2001) · Science · doi:10.1126/science.1060786 · Cited by 768 · cited 1×
“The G93A and C97A mutations that provide the main cold-specific activity boost are located in the single-stranded linker region of the ribozyme between the catalytic core (1-90) and the processivity domain (105-187).”
BackgroundRobertson, M. P., & Joyce, G. F. (2012) · Cold Spring Harbor Perspectives in Biology · doi:10.1101/cshperspect.a003608 · Cited by 556 · cited 1×
“The G93A and C97A mutations that provide the main cold-specific activity boost are located in the single-stranded linker region of the ribozyme between the catalytic core (1-90) and the processivity domain (105-187).”
BackgroundStructurally Complex and Highly Active RNA Ligases Derived from Random RNA Sequences
Ekland, E. H., Szostak, J. W., & Bartel, D. P. (1995) · Science · doi:10.1126/science.7618102 · Cited by 422 · cited 1×
“The G93A and C97A mutations that provide the main cold-specific activity boost are located in the single-stranded linker region of the ribozyme between the catalytic core (1-90) and the processivity domain (105-187).”
BackgroundRibozyme-catalyzed transcription of an active ribozyme
Wochner, A., Attwater, J., Coulson, A., & Holliger, P. (2011) · Science · doi:10.1126/science.1200752 · Cited by 399 · cited 1×
“The G93A and C97A mutations that provide the main cold-specific activity boost are located in the single-stranded linker region of the ribozyme between the catalytic core (1-90) and the processivity domain (105-187).”
BackgroundSelf-citation
Research works printed but not linked: 1
- Atkins, J. F., Gesteland, R. F. & Cech, T. R. (eds) RNA Worlds (Cold Spring Harbor Laboratory, 2011).
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Joyce, G. F., & Szostak, J. W. (2018) · Cold Spring Harbor Perspectives in Biology · doi:10.1101/cshperspect.a034801 · Cited by 308
Damer, B., & Deamer, D. (2015) · Life · doi:10.3390/life5010872 · Cited by 309
Fundamental studies of functional nucleic acids: aptamers, riboswitches, ribozymes and DNAzymes
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Amplification of RNA by an RNA polymerase ribozyme
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“However, the polymerase strongly prefers cytidine-rich templates that lack any secondary structure and has much more limited activity in other contexts (15).”
Background
Metadata
- Authors
- Attwater, J., Wochner, A., & Holliger, P.
- Affiliations
- MRC Laboratory of Molecular Biology (GB)
- Corresponding author
- Holliger, Philipp
- Venue
- Nature Chemistry
- Published
- 2013-10-20
- Volume
- 5
- Issue
- 12
- Pages
- 1011-1018
- ISSN
- 1755-4330 (ISSN-L)
- 1755-4349
- 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
- green
- www.ncbi.nlm.nih.gov
- 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
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