The RNA World

Gianni, Edoardo et al.  “A small polymerase ribozyme that can synthesize itself and its complementary strand.”  Science 391(6789):1022–1028.  Mar 5, 2026.  Section Abstract:  “RNA polymerase ribozymes can replicate RNA, but their large size and structural complexity impede self-replication and preclude their spontaneous emergence.  Here we describe QT45:  a 45-nucleotide polymerase ribozyme, discovered from random sequence pools, that catalyzes general RNA-templated RNA synthesis using trinucleotide triphosphate (triplet) substrates in mildly alkaline eutectic ice.  QT45 can synthesize both its complementary strand using a random triplet pool at 94.1% per-nucleotide fidelity, and a copy of itself using defined substrates, both with yields of ~0.2% in 72 days.”  A laboratory study selected RNA polymerase activity from random-sequence pools, then tested QT45 with triplet substrates in mildly alkaline eutectic ice.  It found that QT45 could synthesize its complementary strand and a copy of itself in separate low-yield reactions; the article reports this result while emphasizing that the reactions did not yet form a self-sustaining cycle. ↩ ↩ ↩ ↩

1. 1a.

   Section Results:  The complementary strand yield is 0.24% from a random 64-triplet pool; the self-copy yield is 0.17% from defined substrates plus one pre-made hexamer, each over 72 days.  The authors state the fidelity consequence and, in a separate sentence, the separation of the two reactions.  “This analysis suggests that at the fidelities observed for QT45 products (92.6% and 94.1%) the population of active variants (quasispecies) would be minimal (<0.5%), but an increase in fidelity to levels observed in other polymerase ribozymes (e.g. 97.4%), would enable maintenance of a sizeable population of active variants of QT45 length in the current mutational landscape (~12% of the total population).”  “These results show that the two key synthetic reactions required for a self-replication cycle:  (−) and (+) strand self-synthesis, can both be carried out by the QT45 ribozyme in separate reactions under identical buffer, salt, and RNA concentrations.” ↩ ↩ ↩

2. 1b.

   Section Results:  “We conjecture that, at higher concentrations, the (+) strand outcompetes triplet substrates for binding to the (−) strand template, driving increasing formation of the unproductive (+)(−) strand duplex.  Indeed, in the absence of the QT45 (+) strand, we observed efficient full-length synthesis by 5TU.  This suggests that the strand-inhibition problem, caused by the formation of the dead end (+)(−) strand duplex, is a key obstacle to closing the self-replication cycle.” ↩

3. 1c.

   Section Results:  “Using the same conditions, we tested QT45 for replication of a model double-stranded RNA template and show that QT45 can copy two complementary RNA sequences simultaneously in a single 'one-pot' reaction using pH-freeze-thaw cycles (fig. S31).”  The conditions were low RNA and magnesium concentrations under iterative coupled pH/freeze-thaw cycling.  The template was a model duplex, not QT45's own plus and minus strands; this remains a one-pot result relevant to an honest summary of the paper. ↩

4. 1d.

   Section Introduction:  This introduction passage states the field's central paradox in the authors' own words.  “However, the large size of these ribozymes challenges the validity of the RNA World hypothesis, due to the implausibility of their spontaneous emergence.  Furthermore, RNA sequences of the size of existing polymerase ribozymes are far outside the range of RNA oligomers observed to form abiotically.  This leads to seemingly paradoxical requirements for RNA self-replication, whereby ribozymes must be large and complex to encode polymerase activity, but that same size and complexity impede both their self-replication and initial emergence.” ↩ ↩

5. 1e.

   Section Introduction:  “However, while these polymerase ribozymes show how RNA can fulfil many of the functions required for self-replication, they have so far fallen short of self-replication, unable to synthesize even their (+) or (−) strands individually.  This may be due to both their size (~150-300 nucleotides), which imposes a high synthetic burden, and their structural complexity, as stably folded RNA can pose a significant obstacle to replication.” ↩

6. 1f.

   Section Results:  The separate-reaction qualification is the authors' own wording and is repeated because it is the central qualification on this result. ↩

7. 1g.

   Section Results:  The quasispecies figures are the authors' model output, not this article's calculation. ↩ ↩

8. 1h.

   Section Results:  The abundance estimate for the QT fold in random sequence is 9.5 × 10^-17.  QT45 was found from pools totalling about 1.2 × 10¹³ sequences after eleven selection rounds plus seven more after mutagenesis.  “a sharp fitness peak with most mutations being detrimental to activity”.  The mutational analysis describes a harsher landscape than the larger 5TU ribozyme, with single mutants ranging from −11.0 to 1.6 in fitness versus −4.6 to 1.6. ↩ ↩ ↩ ↩

9. 1i.

   Section Discussion:  “These syntheses are currently slow and low-yielding, with further improvements in synthetic efficiency and fidelity likely required to overcome chemical degradation and achieve self-sustained replication.  However, the QT ribozyme has only undergone a total of 18 rounds of evolution from a random sequence pool, indicating a likely potential for further development.”  QT45's own half-life under the reaction conditions is about 117 days, against about 16 days for the 200-nucleotide R18. ↩

10. 1j.

    Section Results:  The terminal triplet shows reduced fidelity.  “consistent with a previously described purifying effect of stalling of synthesis caused by misincorporation, leading to an improved overall fidelity of full-length products at the expense of synthetic yield.”  Tjhung and colleagues measured the same trade-off directly in 2020. ↩

11. 1k.

    Section Results:  “To ease the adaptive demands and isolate the shortest motif possible, we leveraged our past observations that RNA polymerase activity is boosted in eutectic ice, due to its capacity to both stabilize polymerase ribozymes and concentrate their substrates.” ↩

12. 1l.

    Figure 4, legend:  “Reaction conditions for both syntheses: 8 nM primer BCy3P10, 8 nM template (t4psP10QT45 or t4msP10QT45), 80 nM ribozyme QT45 (pQT45 for (+) strand) or 5TU+t1.5, 50 nM each substrate, 0.01% Tween 20, 0.4 mM MgCl2, 1.2 mM KCl, 1 mM CHES-KOH, pH 9, incubated for 72 days at -7 °C frozen.” ↩

Urquhart, James.  “Self-synthesising RNA strand offers clues to life's origins.”  Chemistry World (Royal Society of Chemistry).  Feb 16, 2026.  A news article in Chemistry World reports on the QT45 study through comments from lead author Edoardo Gianni and ribozyme researcher David Lilley.  It records Gianni's distinction between separate strand-synthesis reactions and a completed replication cycle, alongside judgments that the work is slow but a step toward proof of principle.  The article was corrected on 17 and 19 February.

1. 2a.

   Edoardo Gianni, the study's lead author, is speaking.  “The two reactions don't happen sequentially or in one pot so we are not yet at the stage of self-replication…  What we describe is self-synthesis – the capacity of an RNA to copy both itself, and synthesise its encoding complementary strand.  Neither of these reactions had been previously demonstrated by a polymerase ribozyme.”  “It substantially lowers the bar that needs to be achieved non-enzymatically before ribozyme-catalysed replication kicks in…  If this is the path needed for life to emerge, this in turn affects the overall probability of life spontaneously emerging from pure chemistry, which we can estimate to be higher than before.” ↩

2. 2b.

   David Lilley, a molecular biologist working on ribozymes at the University of Dundee, gives both assessments.  “That is unbelievably slow…  But this is a good step down the road to proof of principle, showing a critical element required for the plausibility of the RNA world hypothesis.” ↩

3. 2c.

   Gianni's full sentence continues through this optimistic estimate; he also states the team's next goal is to run the two reactions in one pot and close the cycle.  “If this is the path needed for life to emerge, this in turn affects the overall probability of life spontaneously emerging from pure chemistry, which we can estimate to be higher than before.” ↩

Attwater, James et al.  “Trinucleotide substrates under pH-freeze-thaw cycles enable open-ended exponential RNA replication by a polymerase ribozyme.”  Nature Chemistry 17(7):1129–1137.  2025.  Section Results:  “'fragmentary' self-replication”.  A laboratory experiment combined trinucleotide substrates with coupled pH and freeze-thaw cycling to address RNA-duplex strand separation.  It produced exponential replication of both strands of a 29-nucleotide ribozyme-core fragment at up to 1.26-fold per cycle.  Read the word "part" carefully: what replicates exponentially is a segment of the ribozyme, not the whole of it; the authors call it 'fragmentary' self-replication in their own scare quotes.  The article cites it as the one case where a replication cycle has been closed, on 29 nucleotides of about 180. ↩ ↩

1. 3a.

   Page 1129:  “High temperatures degrade RNA templates and ribozyme catalysts, particularly in the presence of divalent cations such as Mg2+ (which boost ribozyme activity but accelerate RNA fragmentation by transesterification).” ↩

2. 3b.

   Page 1131:  “However, the preferred conditions required for strand separation and RNA synthesis are diametrically opposed.  Effective RNA duplex denaturation requires low pH and elevated temperatures to weaken base-pairing interactions, together with low Mg2+ concentrations ([Mg2+]) to reduce duplex stability and minimize RNA hydrolysis, and low RNA strand concentrations to slow down reannealing.  In contrast, optimal RNA synthesis requires a mildly basic pH, high [Mg2+], high ribozyme and triplet substrate concentrations ([RNA]) and ambient to low temperatures.”  “freezing can drive a more than 200-fold concentration of solutes and reduction of water activity within the eutectic brine phase”. ↩ ↩

3. 3c.

   Section Methods, Replication cycling:  “cycling was begun with an initial denaturing step where 0.75 µl of 0.1 M HCl was added (+0.6 mM in the reaction, overwhelming the CHES buffer), the reaction was vortexed, and then incubated for 2 min on a thermocycler preheated to 80 °C.  Next, 0.75 µl of 0.1 M KOH was added, and the reaction was briefly vortexed, then plunged in liquid N2 for 20 s to flash-freeze, before incubation at −7 °C for ~24 h.  Another cycle was initiated by thawing the reactions at room temperature before HCl addition, and so on.” ↩

4. 3d.

   Page 1136:  “Although we had not set out to investigate prebiotic RNA replication scenarios, we note that our RNA replication strategy is general and robust over a range of temperature and pH values (Extended Data Figs. 2 and 6), including flash-freezing, present in modern geothermal fields.” ↩

Orgel, Leslie E.  “Prebiotic Chemistry and the Origin of the RNA World.”  Critical Reviews in Biochemistry and Molecular Biology 39(2):99–123.  2004.  Page 100:  “From our discussion of prebiotic chemistry we will conclude that the abiotic synthesis of RNA is so difficult that it is unclear that the RNA World could have evolved de novo on the primitive Earth, a conclusion that was first emphasized by Cairns-Smith.”  From the introduction.  A review surveys prebiotic nucleotide synthesis, nonenzymatic polynucleotide synthesis and copying, ribozyme catalysts, and prebiotic membranes.  Orgel describes unresolved problems in the RNA-world scenario; his "RNA Later" argument proposes a simpler genetic polymer that preceded RNA and handed off to it, the genetic-takeover model associated with Cairns-Smith.  The article uses this to show that Orgel argues within naturalistic origin-of-life research, not against it. ↩

1. 4a.

   Page 105:  “The inevitable conclusion of this survey of nucleotide synthesis is that there is at present no convincing, prebiotic total synthesis of any of the nucleotides.  Many individual steps that might have contributed to the formation of nucleotides on the primitive Earth have been demonstrated, but few of the reactions give high yields of products, and those that do tend to produce complex mixtures of products.”  This closes the nucleotide-synthesis section.  It was written five years before Powner, Gerland and Sutherland 2009, which bypassed the free-sugar-plus-free-base route entirely; quoting this sentence today without that date is not honest. ↩

2. 4b.

   Page 114:  “Of course, there are still a few problems in prebiotic chemistry that must be solved before the Dream can be turned into a convincing theory!”  The surrounding passage sets out the RNA-first story step by step.  The exclamation mark is Orgel's; he coined the phrase "the Molecular Biologists' Dream" with Joyce in 1999. ↩

3. 4c.

   Page 115:  “It is possible that all of these, and many other difficulties will one day be overcome and that a convincing prebiotic synthesis of RNA will become available.  However, many researchers in the field, myself included, think that this is unlikely and that there must be a different kind of solution to the problem of the origin of the RNA World.”  This opens the "RNA Later" section and is the sentence closest in force to the misattributed one. ↩ ↩

4. 4d.

   Page 118:  “Enough is already known to suggest that each of the steps needed to evolve from a library of randomly sequenced double-stranded RNAs to a self-sustaining RNA organism can be demonstrated in laboratory experiments.”  This balancing sentence belongs with the same summary; page 118 is the last sentence of the body. ↩

5. 4e.

   The word "miracle" does not appear in Orgel's 2004 review.  This is a check anyone can repeat:  a search of the paper's full text for "mirac" finds nothing, and the same search finds every other quotation in this article in its source.  Citing "a near miracle" to that paper attributes a phrase its author did not use. ↩ ↩

Robertson, Michael P., and Gerald F. Joyce.  “The Origins of the RNA World.”  Cold Spring Harbor Perspectives in Biology 4(5):a003608.  2012.  “Previous versions of this article, which were published in the First (1993), Second (1999), and Third (2006) Editions of The RNA World, were coauthored by Leslie Orgel, who died on October 27, 2007.  Many portions of the text have not been changed in the current edition because they remain an accurate reflection of current scientific understanding.”  “I have argued that the self-organization of the reductive citric acid cycle without the help of 'informational' catalysts would be a near miracle”.  A review compares RNA-first and RNA-later models, finds strong evidence for an RNA world but says whether life began with RNA remains unsettled.  The acknowledgments establish the lineage: the 2012 descendant carries forward Joyce and Orgel's earlier chapter in The RNA World (Cold Spring Harbor Laboratory Press), with editions in 1993, 1999, and 2006, but is not a faithful republication of this passage.  Orgel's separate, genuine "near miracle" concerns the reductive citric acid cycle, not RNA (PNAS 97 (2000): 12503–12507, p. 12506); the supposed RNA wording appears nowhere in his 2004 review in Critical Reviews in Biochemistry and Molecular Biology 39:99–123.  The article cites it as the true home of the "near miracle" sentence, where it describes what one of two camps believes. ↩ ↩

1. 5a.

   Section 3.1, page 15:  “Scientists interested in the origins of life seem to divide neatly into two classes.  The first, usually but not always molecular biologists, believe that RNA must have been the first replicating molecule and that chemists are exaggerating the difficulties of nucleotide synthesis.  They believe that a few more striking chemical 'surprises' will establish that a pool of racemic mononucleotides could have formed on the primitive Earth, and that further experiments with different activating groups, minerals, and chiral amplification processes will solve the enantiomeric cross-inhibition problem.  The second group of scientists are much more pessimistic.  They believe that the de novo appearance of oligonucleotides on the abiotic Earth would have been a near miracle.  Time will tell which is correct.”  The 2012 text reads "on the abiotic Earth"; renderings in circulation say "on the primitive earth."  The earlier book editions may have used the earlier wording, and the old note says this could not be checked without opening them.  The 1993 and 1999 book editions print the parenthetical "(The authors subscribe to this latter view.)" immediately after that sentence, but the 2012 text omits it while rewriting the surrounding sentences. ↩

Tjhung, Katrina F. et al.  “An RNA polymerase ribozyme that synthesizes its own ancestor.”  Proceedings of the National Academy of Sciences 117(6):2906–2913.  2020.  A laboratory study from Joyce's group used in-vitro evolution to produce an RNA polymerase ribozyme that synthesises its own ancestor, the class I ligase, as three separate RNA strands.  Only a very small fraction of the assembled ligases retain catalytic activity because of disabling mutations; in the paper announcing this result, the authors describe limits of their own line of work, which the article uses to assess RNA self-copying. ↩ ↩

1. 6a.

   “For any realistic values for selective advantage and per-nucleotide fidelity, the maximum length of RNA that can be maintained is approximately the inverse of the fidelity.  The class I ligase contains approximately 100 nucleotides and thus must be copied with an average fidelity of >99%.  The 38-6 polymerase contains nearly 200 nucleotides and thus must be copied with an average fidelity of >99.5%.”  A gloss is needed or a chemist will catch it: "the inverse of the fidelity" is loose wording for the inverse of the error rate, as the authors' two figures confirm: 1/(1 − 0.99) = 100 and 1/(1 − 0.995) = 200. ↩

2. 6b.

   Section Discussion:  “To achieve self-sustained replication, an RNA polymerase ribozyme must be able to synthesize copies of both itself and its complement in sufficient yields to enable exponential growth.  Although various ribozymes have been shown to copy themselves either partially or entirely, these reactions have always depended on carefully curated sets of preformed oligonucleotide substrates, thereby restricting the ability of the copied RNA to mutate and evolve.  Another issue that has not been addressed in previous studies, or in the current study, is the need to separate the template and product strands, other than by means such as thermal or chemical denaturation.” ↩ ↩ ↩

3. 6c.

   “The hammerhead is synthesized in an ∼2% yield by the 24-3 polymerase after 24 h and in the same yield by the 38-6 polymerase after 1 h, with the products of these two reactions having similar specific activity.  However, if the reaction with the 38-6 polymerase is allowed to continue for 24 h or 72 h, by which time the yield has increased to 20% or 28%, respectively, then the specific activity of the resulting materials decreases significantly.  Thus, it appears that at longer incubation times, the 38-6 polymerase is able to continue the extension of partial-length products that contain a [mismatch].”  “only 83% per nucleotide position”.  “in some cases exceeding a frequency of 40%.”  This is the yield-versus-activity reversal.  The measured fragment yields were 1.1%, 1.0% and 5.7% over three days, with insertions at 1.7% per position; fidelity on the hardest fragment was only 83% per nucleotide position, and substitution and deletion hotspots occurred in some cases at frequencies exceeding 40%. ↩ ↩ ↩

4. 6d.

   “The smaller size of the fragments compared with the contiguous molecule makes them easier to synthesize but does not alleviate the error threshold, which applies to the total number of nucleotides necessary to produce the replicating entity.”  This closing argument closes the fragmentation escape hatch. ↩

Papastavrou, Nikolaos et al.  “RNA-catalyzed evolution of catalytic RNA.”  Proceedings of the National Academy of Sciences 121(11):e2321592121.  2024.  “The 71-89 polymerase contains 182 nucleotides, which would correspond to a maximum permissible error rate of ~1/182 = 0.5% per nucleotide for a complete cycle of plus- and minus-strand synthesis.  However, as with the hammerhead, the error threshold would be much more permissive if selection stringency is high and the cooperative folded structure of the polymerase renders it tolerant to mutation at a significant fraction of the nucleotide positions.  Under suitable selection conditions, a replication fidelity of 97 to 98% would likely be sufficient to enable the RNA-catalyzed evolution of the polymerase ribozyme itself.  Although it is difficult to predict the future path of evolution, both in nature and in directed evolution experiments, such an increase in fidelity does not seem unattainable.”  “the maximum permissible error rate can be at least sevenfold higher than simply the inverse of sequence length.”  “Attempts to recreate RNA-based life in the laboratory must achieve further improvements in replication fidelity to enable the fully autonomous Darwinian evolution of RNA enzymes as complex as the polymerase itself.”  An experimental study from Gerald Joyce's laboratory compared a high-fidelity polymerase ribozyme with a prior low-fidelity variant in parallel lineages.  Across eight rounds of RNA copying and selection, the high-fidelity lineage evolved hammerhead variants with improved fitness while the low-fidelity lineage lost function; the article uses this main passage as evidence of Darwinian evolution on RNA alone and cites the authors' stated fidelity limit.  The closing sentence of the abstract belongs beside that passage and adds that further fidelity improvement is needed for autonomous evolution of polymerases. ↩ ↩

1. 7a.

   “Two evolutionary lineages were carried out in parallel, using either the prior low-fidelity or the newer high-fidelity polymerase.  The former lineage quickly lost hammerhead functionality as the population diverged toward random sequences, whereas the latter evolved new hammerhead variants with improved fitness compared to the starting RNA.”  “Attempts to recreate RNA-based life in the laboratory must achieve further improvements in replication fidelity to enable the fully autonomous Darwinian evolution of RNA enzymes as complex as the polymerase itself.”  “in 18% and 23% yield, respectively after 24 h”.  “yields 11% HHR+ and 7% HHR– after 24 h.”  The measured fidelities are 52-2, 85.6% single pass and 81.4% reciprocal; 71-89, 90.9% and 89.1%. ↩

2. 7b.

   Page 9:  “experimentally determined fitness landscapes for other RNA-cleaving ribozymes suggest that the maximum permissible error rate can be at least sevenfold higher than simply the inverse of sequence length…  In addition, the cooperative nature of RNA folding ensures that many mutations within the catalytic motif have little or no deleterious effect.  The 27-nucleotide region of the hammerhead that was subject to reciprocal copying contains only 10 nucleotides that are completely intolerant of mutation.”  “Under suitable selection conditions, a replication fidelity of 97 to 98% would likely be sufficient to enable the RNA-catalyzed evolution of the polymerase ribozyme itself.  Although it is difficult to predict the future path of evolution, both in nature and in directed evolution experiments, such an increase in fidelity does not seem unattainable.”  This is the paragraph that calibrates the whole argument, and it is from Joyce's own laboratory. ↩

3. 7c.

   Page 2:  “The 52-2 polymerase is capable of synthesizing the hammerhead ribozyme in the presence of either 200 or 50 mM Mg2+, but the higher concentration is required for good yield and the lower concentration is required for good fidelity.”  Page 4:  “For 52-2, the reactions require 200 mM Mg2+ and generate HHR+ and HHR– in 18% and 23% yield, respectively after 24 h.  The 71-89 polymerase, which was evolved to operate with higher fidelity in the presence of 50 mM Mg2+, yields 11% HHR+ and 7% HHR– after 24 h.”  “In contrast, the 71-89 polymerase (in 50 mM Mg2+) synthesizes HHR– with 90.9% single-pass fidelity, and carries out reciprocal synthesis of HHR– and HHR+ with 89.1% overall fidelity”.  HHR+ and HHR– are the hammerhead ribozyme and its complementary strand;  "reciprocal synthesis" is a full copying cycle through both strands.  On the same template the 52-2 polymerase's full-cycle fidelity is 81.4%. ↩ ↩ ↩ ↩

Attwater, James et al.  “Ice as a protocellular medium for RNA replication.”  Nature Communications 1:76.  Sep 21, 2010.  A laboratory study from Philipp Holliger's group at the MRC Laboratory of Molecular Biology in Cambridge.  It ran the R18 polymerase ribozyme in water ice at −7 °C and in solution at 17 °C, measured how fast the ribozyme decayed, how long its products grew, and how accurately it copied, by sequencing more than 3,000 products.  Ice slowed the ribozyme's breakdown and concentrated its substrates.  The article cites it for the measured half-lives that explain why polymerase-ribozyme experiments are run frozen.

1. 8a.

   Page 3, right column:  “The dependence of the RNA polymerase ribozyme on high Mg2+ concentrations for optimal activity accelerates the hydrolysis of its RNA backbone, limiting its half-life to < 52 h at 17 °C.  However, at − 7 °C in ice, its half-life was increased to > 16 days”.  “We found that, whereas ribozyme activity at ambient temperatures ceased after a few days (extending a primer by up to 16 nucleotides), ribozyme polymerase activity in ice continued for weeks, yielding extension products 23 nucleotides long after 25 days.” ↩ ↩

2. 8b.

   Page 1, abstract:  “Ice furthermore relieves the dependence of RNA replication on prebiotically implausible substrate concentrations, while providing quasicellular compartmentalization within the intricate microstructure of the eutectic phase.” ↩

Benner, Steven A.  “Paradoxes in the Origin of Life.”  Origins of Life and Evolution of Biospheres 44:339–343.  2014.  A short commentary by Steven Benner, a prebiotic chemist who works on RNA chemistry, given at the Open Questions on the Origin of Life conference in Kyoto in July 2014.  It sets out five paradoxes, pairs of statements each grounded in theory and observation that together suggest the origins problem cannot be solved, and ends by saying he expects most of them to be resolved.  The article cites the water and probability paradoxes, which bear directly on an RNA world, and his closing expectation with them.

1. 9a.

   Page 342:  “The Water Paradox: Water is commonly viewed as essential for life, and theories of water are well known to support this as a requirement.  So are biopolymers, like RNA, DNA, and proteins.  However, these biopolymers are corroded by water.  For example, the hydrolytic deamination of DNA and RNA nucleobases is rapid and irreversible, as is the base-catalyzed cleavage of RNA in water.  This allows us to construct a paradox: RNA requires water to function, but RNA cannot emerge in water, and does not persist in water without repair.” ↩ ↩

2. 9b.

   Pages 342–343:  “However, experiments show that RNA molecules that catalyze the destruction of RNA are more likely to arise in a pool of random (with respect to fitness) sequences than RNA molecules that catalyze the replication of RNA, with or without imperfections.  Chemical theory expects this to be the case, as the base catalyzed cleavage of RNA is an 'easy' reaction (stereoelectronically), while the SN2 reaction that synthesizes a phosphodiester bond is a 'difficult' reaction.” ↩

3. 9c.

   Page 343:  The paper's final paragraph.  “The prescription that experimentalists focus on paradoxes in origins rather than puzzles in origins (Cleland and Chyba 2002) offers important constraints to researchers who must live in a world where financial resources are limited.  However, the concept of 'paradox' should not be daunting.  We expect that (hope that?) most of these paradoxes will be resolved by experiments that show that the theories that generate them are incorrect, incomplete, or inapplicable to particular molecular systems that just happen to have been present on early Earth.  Others may be resolved because, like most exercises that apply logic to the real world, their reasoning is imperfect.  However, they must be resolved as a condition for the community to conclude that the origins problem has been 'solved'.” ↩

Meyer, Stephen C.  Signature in the Cell:  DNA and the Evidence for Intelligent Design.  HarperOne.  2009.  The design movement's fullest book on the origin of life.  Chapter 14, "The RNA World," sets out five problems with the hypothesis, cites the ribozyme results available in 2009, and argues that ribozyme engineering puts the experimenter's intelligence into the result.  The article quotes Meyer's own statement of the design case and tests his 2009 expectations against the experiments that followed.

1. 10a.

   Page 300:  “My critique details five crucial problems with the RNA world, culminating in a discussion of the information problem.” ↩

2. 10b.

   Page 313:  “Although no one has yet produced a fully self-replicating RNA molecule, some researchers have engineered a molecule that can copy a part of itself—though only about 10 percent of itself and then only if complementary primer strand is provided to the ribozyme by the investigator.” ↩

3. 10c.

   Page 314:  “He estimates that getting a ligase capable of performing the other functions that polymerases must perform—'proper template binding, fidelity and strand separation'—may require between 200 and 300 nucleotides.”  “It may be as low as 189 bases, but it may be much higher, or it may simply be impossible.  Moreover, the problem may be more basic than length.  RNA, with its limited alphabet of four bases, may not even have the capacity to form the complex three-dimensional shapes and distributions of charge necessary to perform polymerase or replicase function.”  "He" is Jack Szostak.  The 189 bases are the length of the partial self-copier Meyer describes on page 313. ↩ ↩

4. 10d.

   Page 315:  “Given this and the experiments mentioned above showing the rarity of functional ribozymes (to say nothing of polymerases) within RNA sequence space, the odds of a functional, self-replicating RNA sequence arising by chance are exceedingly small.” ↩

5. 10e.

   Pages 316–317:  “the transition from the RNA-based translation system to the current protein-based translation system would have required at some point the production of more than 100 different proteins”. ↩

6. 10f.

   Page 320:  “Szostak and his colleagues function as the replicators.  They generate the crop of variant sequences.  They make the choices about which of these sequences will survive to undergo another round of directed evolution.  Moreover, they make these choices with the benefit of a foresight that neither natural selection nor any other undirected or unintelligent process can—by definition—possess.” ↩

The article's arithmetic.  One QT fold per 10¹⁷ random sequences, Gianni and colleagues' estimate,¹ means about 10¹⁷ molecules are needed to expect one.  10¹⁷ ÷ 6.02 × 10²³ per mole = 1.7 × 10^-7 mol.  A 45-nucleotide RNA weighs about 45 × 330 = 14,850 grams per mole, so the pool weighs about 2.5 × 10^-3 grams, or 2.5 milligrams.  This prices sequence rarity alone.  It assumes a supply of random 45-nucleotide RNA to sample from, which is the feedstock problem, not the rarity problem. ↩ ↩ ↩

Attwater, James et al.  “Ribozyme-catalysed RNA synthesis using triplet building blocks.”  eLife 7:e35255.  2018.  Section Triphosphorylated triplet synthesis:  “pppNNN was generated by combination of equal amounts of each of the 64 triplet stocks.”  The article calls this a step inherited by the polymerase-ribozyme literature and argues that it is not prebiotic.  An in vitro evolution study produced a mutualistic RNA heterodimer that synthesizes RNA with triphosphorylated trinucleotides and can copy structured templates and segments of its catalytic subunit.  Its triplets were made with T7 RNA polymerase from synthetic DNA templates, purified on a 30% acrylamide/urea gel, and combined in equal amounts from 64 stocks; the article cites this preparation as a dependence on protein-enzyme feedstock and manual mixing. ↩ ↩ ↩

Attwater, James et al.  “In-ice evolution of RNA polymerase ribozyme activity.”  Nature Chemistry 5(12):1011–1018.  2013.  Section Abstract:  “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.”  From the body.  “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 at 17 °C,”.  “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.”  The templates were favourable repeats rather than mixed sequence; at −7 °C the same ribozyme managed only 118 nucleotides, and tC9Y did not copy tC9Y.  An in vitro evolution experiment used compartmentalized bead-tagging to select variants from a 5×10^7-member mutant library of the R18 RNA polymerase ribozyme.  After eight selection rounds, tC9Y synthesized RNA up to 206 nucleotides long on favorable templates, including products longer than itself; the article uses it as a length milestone while stressing that long-product synthesis is not self-copying. ↩

1. 13a.

   Section Synthesis of long RNAs in ice and at ambient temperatures:  “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”.  “the Y mutations conferred a modest fidelity improvement at 17°C to 98.3% compared to 97.3% for tC19, and in ice to 94.8% compared to 93.4% for R18.”  tC19 is the parent polymerase;  R18 is the original 2001 polymerase. ↩

2. 13b.

   Section Materials and methods:  “NTPs were purchased from Promega and mutagenic dNTPs from TriLink.” ↩

Horning, David P., and Gerald F. Joyce.  “Amplification of RNA by an RNA polymerase ribozyme.”  Proceedings of the National Academy of Sciences 113(35):9786–9791.  2016.  Section Abstract:  “The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA.  Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR.  Thus, the two prerequisites of Darwinian life—the replication of genetic information and its conversion into functional molecules—can now be accomplished with RNA in the complete absence of proteins.”  The tRNA was made in low yield, the exponentially amplified templates were short compared with the approximately 180-nucleotide polymerase, and the experimenter supplied the thermal cycling.  An in vitro evolution study selected an improved polymerase ribozyme through 24 rounds, beginning with a population of 10^14 variants and selecting for functional aptamer completion.  The evolved ribozyme made structured RNAs and amplified short RNA templates by more than 10,000-fold in an RNA-catalyzed form of PCR; the article uses it as evidence for RNA-only amplification while noting that its templates were short and thermal cycling was supplied by the experimenter. ↩

The article's arithmetic.  A 117-day half-life leaves 2^-72/117 ≈ 0.65 of the ribozyme after 72 days, so about 35% decays.¹  Over the same 72 days 0.17% to 0.24% of templates are copied to full length.¹  0.35 ÷ 0.0024 ≈ 145, and 0.35 ÷ 0.0017 ≈ 205.  The two fractions describe different populations, decayed ribozymes and copied templates, so this is an order-of-magnitude comparison and not a growth model.  It applies to ice at −7 °C, where QT45 was measured. ↩

Joyce, Gerald F. et al.  “Chiral selection in poly(C)-directed synthesis of oligo(G).”  Nature 310:602–604.  1984.  Section Abstract:  “We report here that poly(C)-directed oligomerization of activated guanosine mononucleotides proceeds readily if the monomers are of the same optical handedness as the template, and is indeed far less efficient if the monomers are of the opposite handedness.  However, in template-directed reactions with a racemic mixture, monomers of the opposite handedness to the template are incorporated as chain terminators at the 2′(3′) end of the products.  This inhibition raises an important problem for many theories of the origin of life.”  The mechanism matters: the wrong-handed monomer does not simply fail to add; it adds and the chain cannot be extended past it.  Joyce restated the problem thirty years later in Sczepanski, Jonathan T., and Gerald F. Joyce, Nature 515 (2014), pages 440–442, calling it.  “a severe challenge for the spontaneous emergence of RNA-based life.”  A laboratory experiment compared poly(C)-directed copying with activated guanosine mononucleotides of matching and opposite optical handedness.  It found that opposite-handed monomers can be incorporated as chain terminators, which the article cites as a problem for RNA copying in a racemic environment. ↩

Joyce, Gerald F., and Jack W. Szostak.  “Protocells and RNA Self-Replication.”  Cold Spring Harbor Perspectives in Biology 10(9):a034801.  2018.  A review outlines the proposed relationship between RNA heredity and protocells and surveys chemical and RNA-catalyzed replication systems.  It says early RNA copying would need reasonable rate and fidelity, and that protocell growth, division, and genetic replication would need to remain compatible; the article uses it for chirality, strand-separation, and fidelity limits on RNA replication.

1. 17a.

   Section 4.5 Complete Cycles of Replication:  “The inheritance of RNA-based genetic information requires cycles of both plus- and minus-strand synthesis.  Because the product of template copying is a double-stranded RNA, there must be some means of either strand separation or strand displacement synthesis.  Transient temperature fluctuations could lead to thermal strand separation, but long RNA duplexes (≥30 base pairs) are difficult to denature thermally.”  “A more difficult problem is that strand reannealing is very fast relative to the time scale of nonenzymatic template copying.  Strand reannealing can be slowed by dilution but achieving a reannealing time of hours would require strand concentrations in the picomolar range, which likely would be too low to maintain RNA replication within a protocell.” ↩

2. 17b.

   Section 4.5 Complete Cycles of Replication:  “The alternative solution of strand displacement synthesis is appealing because this is the strategy that is universally used in biology.  However, biological strand displacement relies on helicases and/or polymerases that can transduce some of the chemical energy used to drive primer extension into the mechanical energy needed for strand separation.  It is unclear how this could be achieved in a prebiotic setting devoid of enzymes.” ↩

3. 17c.

   Section 4.4 Fidelity and the Error Threshold:  “For simplicity, consider a self-replicating RNA that is formed by n condensation reactions, each having mean fidelity q.  The probability of obtaining a completely error-free copy is given by qⁿ…  If an advantageous individual is to outgrow its competitors, qⁿ must exceed the superiority, s, of that individual.”  “is highly sensitive to the fidelity of replication but depends only weakly on the superiority of the advantageous individual.”  For s > 1 and q > 0.9, their simplified form is n < ln s / (1 − q).  Raising the superiority from twofold to a hundredfold raises the calculated maximum length at 94.1% fidelity from about 12 nucleotides to about 78, which is longer than QT45; a single number from this formula is not a wall. ↩ ↩

4. 17d.

   Section 3.1 RNA as the First Genetic Material:  “This is a serious problem because experiments on the nonenzymatic template-directed polymerization of imidazole-activated mononucleotides suggest that the polymerization of the D-enantiomer is strongly inhibited by the L-enantiomer (Joyce et al. 1984).  This difficulty may not be insuperable;  perhaps with a different mode of phosphate activation, the inhibition would be less severe.  However, enantiomeric cross-inhibition is certainly a serious problem if life arose in a racemic environment.”  Both the caveat and the statement of seriousness are the authors' own and belong together. ↩

5. 17e.

   Section 5.3 From Polymerase to Replicase:  “Generalized RNA-catalyzed RNA replication has not yet been achieved and it is not possible for the class I polymerase ribozyme to synthesize additional copies of itself.  The most advanced form of the polymerase cannot replicate long RNA sequences because attempts to do so are thwarted by the emergence of shorter amplicons that are copied more efficiently…  Despite falling short of the goal of generalized RNA-catalyzed RNA replication, there is one example of an RNA enzyme that catalyzes its own amplification and can undergo limited Darwinian evolution.  This replication system uses a pair of ligase ribozymes that each catalyze the formation of the other, using a mixture of four different substrate oligonucleotides.”  Written in 2018 and superseded in part by Gianni 2026, but the clause about the four pre-made substrates is the one that still stands. ↩ ↩

6. 17f.

   Section 2.1 Pros and Cons of Membrane Systems:  “However, nonenzymatic copying of RNA templates requires very high concentrations of divalent cations, typically Mg2+, and most ribozymes require high concentrations of Mg2+ for optimal activity.  In contrast, fatty acid–based membranes become unstable in ≥4 mM Mg2+, indicating an apparent incompatibility between the two systems.”  “As an added benefit, the Mg2+-catalyzed degradation of RNA is prevented by citrate.  However, it is unlikely that sufficient citrate was present in the prebiotic environment, and thus alternatives must be sought.” ↩

Kun, Ádám et al.  “Real ribozymes suggest a relaxed error threshold.”  Nature Genetics 37(9):1008–1011.  2005.  “The error threshold also depends on the fitness landscape.  In an RNA world, many neutral and compensatory mutations can raise the threshold, below which the functional phenotype, rather than a particular sequence, is still present.  Here we show, on the basis of comparative analysis of two extensively mutagenized ribozymes, that with a copying fidelity of 0.999 per digit per replication the phenotypic error threshold rises well above 7,000 nucleotides, which permits the selective maintenance of a functionally rich riboorganism with a genome of more than 100 different genes, the size of a tRNA.  This requires an order of magnitude of improvement in the accuracy of in vitro-generated polymerase ribozymes.”  A comparative analysis of two extensively mutagenized ribozymes finds that neutral and compensatory mutations can preserve function beyond a sequence-level error threshold.  At 0.999 copying fidelity, the threshold rises above 7,000 nucleotides, roughly seventy times the naive threshold, but reaching it still requires a tenfold accuracy improvement over laboratory polymerases; the article uses this to qualify the naive error-threshold objection. ↩

The article's arithmetic, with the formula as Joyce and Szostak state it:¹⁷ maximum length n = ln s ÷ (1 − q), where s is how much fitter the best sequence is than its mutants.  At q = 0.941, 1 − q = 0.059.  With s = 10, n = 2.30 ÷ 0.059 ≈ 39 nucleotides.  With s = 100, n = 4.61 ÷ 0.059 ≈ 78.  A full cycle copies two strands, so errors from both add:  if the second strand were copied as accurately as the first, the cycle would run at about 0.941 × 0.941 ≈ 88.5%, close to the 89.1% measured for the 71-89 polymerase.⁷ ↩

Kent, Alexandra D. et al.  “A polymerase ribozyme increases copying fidelity through pyrophosphate-mediated RNA repair.”  bioRxiv.  Feb 5, 2026.  This footnote cites the preprint text.  The authors call this repair rather than proofreading, and the difference matters: proofreading checks as a chain is copied, whereas this process rescues a chain that has already stalled.  Published after the article's cutoff, this laboratory study tested pyrophosphate-mediated repair of RNA sequences terminated in a mismatch, a non-extendable 2′-3′ cyclic phosphate, or both.  Under saturating pyrophosphate, a polymerase ribozyme restored terminated strands so copying could continue along extended templates, including the ribozyme itself; the article cites this as RNA-only rescue of stalled chains that can raise copying fidelity.  The published version appears in Proceedings of the National Academy of Sciences 123(23), article e2606892123, DOI 10.1073/pnas.2606892123. ↩

Le Vay, Kristian, and Hannes Mutschler.  “The difficult case of an RNA-only origin of life.”  Emerging Topics in Life Sciences 3(5):469–475.  2019.  A perspective review surveys the chemical and copying problems in RNA-first scenarios and concludes that robust, continuous RNA self-replication from realistic feedstocks has not been demonstrated; it also notes that ribozyme systems have shown only simple ligation or recombination from defined oligonucleotides.  The article says this is reference 22 of Gianni et al. 2026, making the gap one the QT45 authors themselves cite. ↩

Song, Emilie Yeonwha et al.  “Prebiotically Plausible RNA Activation Compatible with Ribozyme-Catalyzed Ligation.”  Angewandte Chemie International Edition 60(6):2952–2957.  2021.  Page 2952.  A laboratory experiment demonstrates DAP and imidazole activation of RNA in frozen water-ice, producing 2′,3′-cyclic phosphate mono- and oligonucleotides usable by hairpin-ribozyme catalysis; it also states that credible non-enzymatic pathways for high-yielding 5′-triphosphate activation are missing.  The article presents the study as showing that prebiotic activation chemistry is not empty while noting that the activated triplet pool remains missing; its accompanying caveat cites Kim and Benner 2021 (Astrobiology 21:298-306), whose abstract reports 2-3% nucleoside 5′-triphosphates with nickel, borate, urea, and cyclic trimetaphosphate under evaporative heating. ↩

Lee, David H. et al.  “A self-replicating peptide.”  Nature 382:525–528.  1996.  “despite the central importance of molecular self-replication in the origin of life, the feasibility of peptide self-replication has not been established experimentally.”  A laboratory experiment reports autocatalytic synthesis of a 32-residue α-helical peptide based on the leucine-zipper domain of the modern yeast transcription factor GCN4, using two supplied fragments in neutral, dilute aqueous solutions.  The authors frame the work as addressing an experimentally unestablished feasibility that matters to origin-of-life research; because the starting material comes from a modern yeast protein and the fragments are supplied, the chemist's-hand objection applies here exactly as it does to RNA systems.  Its parabolic growth, in which completed copies inhibit further production, matters because the article argues that this behavior prevents the faster variant from eliminating the slower one. ↩

1. 23a.

   “The self-replication process displays parabolic growth pattern with the initial rates of product formation correlating with the square-foot of initial template concentration.”  "square-foot" is a typographical error for "square-root" in the indexed abstract, reproduced as printed rather than silently corrected; the chemistry is the square root.  The claim that a parabolic replicator cannot support Darwinian selection between variants is standard in the replication literature but is not stated in this abstract; it is the article's gloss. ↩

Musgrave, Ian.  “Lies, Damned Lies, Statistics, and the Probability of Abiogenesis Calculations.”  The TalkOrigins Archive.  1998.  “absurdly low”.  An essay responding to claims that abiogenesis is statistically impossible uses the 32-amino-acid Ghadiri peptide ligase to calculate a one-trial assembly probability of 1 chance in 4.29 × 10^40, then argues that this probability does not say when the event will occur in a series and considers many simultaneous trials.  The article presents Musgrave's essay as the standard skeptical counterargument; after citing three Discovery Institute pages, it names the opposing case because an article ignoring that counterargument would be arguing with itself, and says its own arithmetic reaches the same verdict on the same arguments by a different route. ↩

Lincoln, Tracey A., and Gerald F. Joyce.  “Self-sustained replication of an RNA enzyme.”  Science 323:1229–1232.  2009.  A laboratory experiment reports two RNA enzymes that catalyze one another's synthesis from a total of four oligonucleotide substrates and self-sustained exponential amplification without proteins or other biological materials, doubling in about an hour.  Both phrases quoted by the article come from the abstract.  The limitation in the article's framing is Joyce and Szostak's later assessment in their 2018 review: this class of cross-replicating ligases joins pre-formed oligonucleotide substrates rather than copying a sequence, a qualification anyone relying on this paper against the article's argument should read. ↩

Robertson, Michael P., and Gerald F. Joyce.  “Highly efficient self-replicating RNA enzymes.”  Chemistry & Biology 21:238–245.  2014.  “its exponential growth rate to 0.14 min(-1), corresponding to a doubling time of 5 min.  This is close to the limit of 0.21 min(-1) imposed by the rate of product release, but sufficient to enable more than 80 logs of growth per day.”  “inventive evolution within this synthetic genetic system depends on the diversity of the evolving population,”.  An experimental report describes directed evolution raising the growth rate of a self-replicating RNA enzyme, while identifying population diversity as a constraint on further evolution.  The article cites the five-minute doubling time as the later improvement over the 2009 system and highlights the authors' own qualification about diversity. ↩

Shapiro, Robert.  “A Simpler Origin for Life.”  Scientific American 296(6):46–53.  Jun 2007.  “No physical law need be broken for spontaneous RNA formation to happen, but the chances against it are so immense, that the suggestion implies that the non-living world had an innate desire to generate RNA.”  “These requirements are general in nature, however, and are immensely more probable than the elaborate multi-step pathways needed to form a molecule that can function as a replicator.”  “A highly implausible start for life, as in the RNA-first scenario, implies a universe in which we are alone…  The small-molecule alternative, however, is in harmony with the views of biologist Stuart Kauffman:  'If this is all true, life is vastly more probable than we have supposed.'”  A Scientific American essay argues that RNA-first faces major difficulties but that Shapiro's small-molecule alternative makes life more probable, not less.  Shapiro was professor emeritus of chemistry at New York University and a specialist in DNA damage; he died in 2011, and the article uses his naturalistic alternative to prevent an apologetic inversion of his thesis.  His closing paragraph says the small-molecule alternative makes life vastly more probable than supposed. ↩

1. 27a.

   “The drying lagoon claim is not unique.  In a similar spirit, other prebiotic chemists have invoked freezing glacial lakes, mountainside freshwater ponds, flowing streams, beaches, dry deserts, volcanic aquifers and the entire global ocean (frozen or warm as needed).”  Shapiro objects to the urea concentration used in Robertson and Miller's 1995 cytosine synthesis (Nature 375:772), which was justified by invoking drying lagoons.  He continues by listing other proposed settings. ↩

2. 27b.

   “The analogy that comes to mind is that of a golfer, who having played a golf ball through an 18-hole course, then assumed that the ball could also play itself around the course in his absence.  He had demonstrated the possibility of the event;  it was only necessary to presume that some combination of natural forces (earthquakes, winds, tornadoes and floods, for example) could produce the same result, given enough time.  No physical law need be broken for spontaneous RNA formation to happen, but the chances against it are so immense, that the suggestion implies that the non-living world had an innate desire to generate RNA.”  The "no physical law need be broken" clause must be quoted with the rest because Shapiro is explicitly declining the second-law and impossibility arguments that apologetics runs; he is not making an impossibility argument. ↩

3. 27c.

   “hordes of defective building blocks, the inclusion of which would ruin the ability of the chain to act as a replicator.  The simplest flawed unit would be a terminator, a component that had only one 'arm' available for connection, rather than the two needed to support further growth of the chain.”  Shapiro says a prebiotic pool would contain defective building blocks and gives the chain-terminator problem in general form. ↩

4. 27d.

   “Over the years, many theoretical papers have advanced particular metabolism first schemes, but relatively little experimental work has been presented in support of them…  They have not yet demonstrated the operation of a complete cycle or its ability to sustain itself and undergo further evolution.  A 'smoking gun' experiment displaying those three features is needed to establish the validity of the small molecule approach.”  “no experimental demonstration has been made.”  The technical version of the same argument is in Quarterly Review of Biology 81 (2006): 105–125, whose abstract likewise concedes the quoted point. ↩

Axe, Douglas D.  “Estimating the prevalence of protein sequences adopting functional enzyme folds.”  Journal of Molecular Biology 341(5):1295–1315.  2004.  A laboratory study by Douglas Axe, then at the Babraham Institute in Cambridge, of how rare working enzyme sequences are.  He randomised clusters of ten side-chains in a beta-lactamase fold in four experiments and found about one in 10⁶⁴ fold-consistent sequences worked, then combined that with two estimated factors to reach a bound of one in 10⁷⁷ for a specific function, the high end of a range that runs to one in 10⁵³.  The article cites it as the design movement's best-known protein rarity figure, to compare with the measured rarity of a working ribozyme.

1. 28a.

   Page 1295, abstract:  “Combined with the estimated prevalence of plausible hydropathic patterns (for any fold) and of relevant folds for particular functions, this implies the overall prevalence of sequences performing a specific function by any domain-sized fold may be as low as 1 in 10⁷⁷, adding to the body of evidence that functional folds require highly extraordinary sequences.” ↩

Tour, James M.  “Much Ado About Nothing.”  Inference: International Review of Science 6(4).  Jan 2022.  A review essay by James Tour, a synthetic organic chemist at Rice University, of a 2021 paper by Jiménez, Gibard and Krishnamurthy that made short DNA chains from purchased deoxynucleosides.  It closes with a footnoted list of problems Tour says the RNA world fails to account for.  The article quotes that list and tests two of its items against measurements.

1. 29a.

   The closing list of problems:  “These notions and the RNA world hypothesis itself have proven remarkably durable, even though they fail to account for … the thermal instability of RNA, which decomposes rapidly, even at 0°C; the fact that even specifically designed and primed RNAs have never been shown to duplicate more than 7% of themselves, and that those segments were found to be too short to serve as new templates”.  Tour's footnote for the instability item is a supplier's web page, Merck's Oligonucleotide Handling & Stability page.  For the 7% item he cites Johnston and colleagues (2001) and Zaher and Unrau (2007). ↩

Futerman, Anthony H.  “A Storm in a Primordial Teacup.”  Inference: International Review of Science 7(1).  Jun 2022.  A letter to the editors from a Weizmann Institute biochemist who works on lipids, the one letter the journal printed in reply to Tour's "Much Ado About Nothing."  He applies Tour's critique to a 2020 enzyme-free lipid synthesis and agrees with it.  Tour's 2017 open letter in the same journal thanks Futerman for information on lipids, which helps weigh the letter as an independent reply.

1. 30a.

   “Just as the Jiménez et al. paper seems to be much ado about nothing, I would argue, agreeing with Tour, that many similar articles about the origin of life do not actually explain anything.” ↩

Jia, Tony Z. et al.  “Retraction: Oligoarginine peptides slow strand annealing and assist non-enzymatic RNA replication.”  Nature Chemistry 9(12):1286.  Nov 23, 2017.  Section Retraction:  “our efforts to repeat and follow up on the results have been unsuccessful,”.  “we now understand that the data reported in the published article are the result of false positives that arose from an incorrectly designed experiment in which random errors, including transfer and concentration errors, affected the ratio of the concentrations of the RNA template and its complementary strand…  The authors would like to thank Dr Tivoli Olsen for her extensive efforts to unravel the errors in our Article and we apologize to the scientific community for any confusion arising from our publication.”  The retracted article appeared in Nature Chemistry 8 (2016), pages 915–921, DOI 10.1038/nchem.2551, and claimed that arginine-rich peptides slowed complementary-strand annealing while permitting primer extension.  “by up to several thousand-fold”.  Jack W. Szostak's quoted public comment is from Adam Marcus, "'Definitely embarrassing:' Nobel Laureate retracts non-reproducible paper in Nature journal," Retraction Watch, 5 December 2017: https://retractionwatch.com/2017/12/05/definitely-embarrassing-nobel-laureate-retracts-non-reproducible-paper-nature-journal/.  “In retrospect, we were totally blinded by our belief [in our findings]…we were not as careful or rigorous as we should have been (and as Tivoli was) in interpreting these experiments.”  “The only saving grace is that we are the ones who discovered and corrected our own errors, and figured out what was going on.”  A retraction notice reports unsuccessful efforts to repeat claims that arginine-rich peptides slowed strand reannealing and enabled RNA copying in the presence of the complementary strand, and attributes the false positives to experimental design and errors in RNA concentration ratios.  The notice is unusually candid.  It says that vesicle stability with arginine-rich peptides and the failure of acidic peptides to condense RNA did reproduce, and thanks Dr Tivoli Olsen for uncovering the errors.  The article uses it to document the failure of a proposed solution to strand separation. ↩ ↩

Kahana, Amit.  “The RNA-First Fallacy: Conflating Evolutionary Ancestry with Prebiotic Primacy.”  Life 16(5):837.  2026.  Published after the article's cutoff, this is an opinion essay distinguishing evolutionary RNA ancestry from prebiotic RNA primacy.  It argues that ribozyme experiments show compatibility with prebiotic conditions rather than probability or necessity, and proposes chemically diverse autocatalytic networks, including lipid assemblies, as more probable precursors.  The article cites both its critique of RNA-first and its alternative proposal. ↩

1. 32a.

   His alternative is lipid assemblies.  “exhibit notable chemical leniency, befitting an autocatalytic set that does not inherently demand any specific molecular identity or notable seeded complexity.  This makes their emergence not merely possible, but statistically far more probable than any consequential ribozyme in a prebiotic context.”  In the same passage, Kahana concedes that.  “experimental evidence for collectively autocatalytic sets in small molecules is still scarce.”  Quoting his criticism of the RNA world without his proposal would be a quote-mine of exactly the kind this article audits elsewhere. ↩