Nonenzymatic copying of RNA templates containing all four letters is catalyzed by activated oligonucleotides
Open accessAbstract
The nonenzymatic replication of RNA is a potential transitional stage between the prebiotic chemistry of nucleotide synthesis and the canonical RNA world in which RNA enzymes (ribozymes) catalyze replication of the RNA genomes of primordial cells. However, the plausibility of nonenzymatic RNA replication is undercut by the lack of a protocell-compatible chemical system capable of copying RNA templates containing all four nucleotides. We show that short 5′-activated oligonucleotides act as catalysts that accelerate primer extension, and allow for the one-pot copying of mixed sequence RNA templates. The fidelity of the primer extension products resulting from the sequential addition of activated monomers, when catalyzed by activated oligomers, is sufficient to sustain a genome long enough to encode active ribozymes. Finally, by immobilizing the primer and template on a bead and adding individual monomers in sequence, we synthesize a significant part of an active hammerhead ribozyme, forging a link between nonenzymatic polymerization and the RNA world.
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Written from the paper’s full text.
TL;DR
Short 5′-activated oligonucleotides act as catalysts that accelerate nonenzymatic primer extension and enable one-pot copying of mixed-sequence RNA templates containing all four letters. The approach achieves 98% fidelity and supports synthesis of a significant portion of an active hammerhead ribozyme.
Key findings
- Activated downstream helper oligonucleotides strongly catalyze primer extension: an activated AGC trimer increased the rate by over two orders of magnitude compared with unactivated trimers, and a single G addition with an activated downstream trinucleotide was about twice as fast as with up to four sequential activated G monomers.
- With activated trimers, extension rates for all four monomers increased by at least two orders of magnitude; the previous at-least-three-orders-of-magnitude difference between A/U and G/C polymerization was reduced to roughly one order of magnitude.
- Fidelity of trimer-assisted polymerization averaged 98%, allowing effective genome sizes of 50 nucleotides; the fastest mismatch, G on a U template, was approximately 5% as fast as A on the same template, while all other mismatches were at least two orders of magnitude slower than matched pairs.
- One-pot copying with all four monomers and four trimers gave ~80% yield of primer extended by four monomers after 16 hr, whereas single-monomer addition reached >95% yield after 10 min; bead-based sequential synthesis of hammerhead HH2 reached 75–95% conversion per step, with full-length HH2 comprising 6% of products, and the crude product catalyzed 50% cleavage of HH1 after 16 hr.
Methods
- Nonenzymatic primer extension used 2-methylimidazole-activated monomers and oligonucleotides on RNA primer/template complexes, with reaction conditions including 200 mM MgCl₂/CHES pH 9 or 100 mM MgCl₂/Tris pH 8, 25–50 mM monomer, and 1–2 mM trimer; rates were calculated as pseudo-first-order constants from denaturing gel electrophoresis.
- Fidelity was assessed by measuring matched and mismatched monomer-template combinations for all four templates, and modified U monomers 2-thiouridine and 2-thioribothymine were tested for rate effects.
- Bead-immobilized reactions attached primer and biotinylated template to streptavidin beads, added matched monomer/trimer pairs sequentially with washes between steps, released product with a DNA complement, treated with DNase, and assayed hammerhead ribozyme cleavage using Cy5-labeled HH1.
Limitations
- The paper states that the method can only produce sequences >7 nucleotides in one reaction in low yields due to cross-inhibitory effects of trimers with overlapping binding sites, and that further advances are required for copying longer functional RNA sequences in one-pot, protocell-compatible conditions.
- The paper states that a plausible source of chemical energy to re-activate monomers and oligomers after hydrolytic loss of the leaving group is a key missing component, and that more rigorous fidelity tests with competing monomers in all sequence contexts are ongoing.
- Evident from the text: the bead-based synthesis required 150 hr total, with individual steps varying from 30 min to 22 hr; full-length HH2 was only 6% of reaction products; the reason for rate discrepancies between bead and solution reactions is unknown; and no 2-thiouridine monomers were used in the ribozyme synthesis to protect catalytic capacity.
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Claims the paper makes
0 of 28 cited sources checked · 0 supported · 0 flagged
- Findingown data
At subsaturating concentrations of helper oligonucleotide, the rate of the primer extension reaction increased with increasing concentrations of di- or tri-nucleotide helper.
“At subsaturating concentrations of helper oligonucleotide, the rate of the primer extension reaction increased with increasing concentrations of di- or tri-nucleotide helper (Figure 2—figure supplement 1).”
Quote in the paperNot a substantial claim - Findingown data
When the same AGC trimer was activated as a 5′-phosphoro-2-methylimidazolide and used as a downstream helper oligonucleotide, the rate of primer extension by addition of a G monomer was increased by over two orders of magnitude compared to reactions with unactivated trimers.
“In contrast, when the same AGC trimer was activated as a 5′-phosphoro-2-methylimidazolide and used as a downstream helper oligonucleotide, the rate of primer extension (by addition of a G monomer) was increased by over two orders of magnitude (Figure 1c–f, reaction 6) compared to reactions with unactivated trimers.”
Quote in the paperSubstantial claim - Findingown data
The maximum rate of the trimer-assisted reaction was significantly faster than that of the dimer-assisted reaction.
“Because the maximum rate of the trimer-assisted reaction was significantly faster than that of the dimer-assisted reaction, we proceeded with trimers as the downstream helper oligonucleotides for the remainder of this study.”
Quote in the paperSubstantial claim - Findingown data
Binding of an unactivated AGC trimer downstream of an activated G monomer conferred only a modest increase in the rate of addition of the G monomer to the primer.
“The binding of an unactivated AGC trimer, with either a 5′ hydroxyl or a 5′ phosphate, downstream of an activated G monomer conferred only a modest increase in the rate of addition of the G monomer to the primer (Figure 1c–f, reactions 4 and 5), consistent with previous reports of unactivated downstream ‘helper’ oligonucleotides (Deck et al., 2011; Jauker et al., 2015; Kervio et al., 2010).”
Quote in the paperNot a substantial claim0 of 3 sources checked - Findingown data
The primer extension reaction proceeded almost 100 times more rapidly than the ligation reaction.
“To our surprise, the primer extension reaction proceeded almost 100 times more rapidly than the ligation reaction (Figure 1d).”
Quote in the paperSubstantial claim
- Findingown data
At saturating concentrations of helper oligonucleotides, the rate of primer extension improved as the length of the helper increased from mononucleotide to dinucleotide to trinucleotide, and then stayed approximately constant with the tetranucleotide.
“At saturating concentrations (Figure 2—figure supplement 1) of each of the oligonucleotides, the rate of primer extension improved as the length of the helper increased from mononucleotide to dinucleotide to trinucleotide, and then stayed approximately constant with the tetranucleotide (Figure 2a).”
Quote in the paperSubstantial claim - Findingown data
Activated helper oligonucleotides facilitate the addition of all four RNA monomers individually and in sequence in a single reaction.
“Activated helper oligonucleotides facilitate the addition of all four RNA monomers individually and in sequence in a single reaction.”
Quote in the paperSubstantial claim - Findingown data
Short 5′-activated oligonucleotides allow for the one-pot copying of mixed sequence RNA templates.
“and allow for the one-pot copying of mixed sequence RNA templates”
- Also stated in: § Results · plain
“Adding two, three or four trimers together resulted in the generation of primers extended by two, three or four monomers respectively (Figure 4b).”
Quote in the paperSubstantial claim - Also stated in: § Results · plain
- Findingown data
The rate of primer extension with a single G in the presence of an activated downstream trinucleotide was about twice as fast as the rate when the primer was followed by up to four sequential activated G monomers.
“The rate of primer extension with a single G in the presence of an activated downstream trinucleotide was about twice as fast as the rate when the primer was followed by up to four sequential activated G monomers (Figure 1c–f, reaction 1 and 6).”
Quote in the paperNot a substantial claim - Findingown data
By immobilizing the primer and template on a bead and adding individual monomers in sequence, the authors synthesized a significant part of an active hammerhead ribozyme.
“Finally, by immobilizing the primer and template on a bead and adding individual monomers in sequence, we synthesize a significant part of an active hammerhead ribozyme, forging a link between nonenzymatic polymerization and the RNA world.”
- Also stated in: § Introduction · plain
“Finally, when a template is immobilized by attachment to a bead, sequential addition of activated monomers and helper oligonucleotides successfully promoted the synthesis of a significant part of an active hammerhead ribozyme.”
Quote in the paperSubstantial claim - Also stated in: § Introduction · plain
- Findingown data
Short 5′-activated oligonucleotides act as catalysts that accelerate primer extension.
“We show that short 5′-activated oligonucleotides act as catalysts that accelerate primer extension, and allow for the one-pot copying of mixed sequence RNA templates.”
- Also stated in: § Introduction · plain
“Here we show that the presence of a leaving group at the 5′ end of a helper oligonucleotide can be a source of significant catalysis.”
Quote in the paperSubstantial claim - Also stated in: § Introduction · plain
- Findingown data
The rate of trimer assisted primer extension with a single monomer decreased in the presence of additional overlapping downstream trimers.
“This hypothesis is supported by the decreased rate of trimer assisted primer extension with a single monomer in the presence of additional overlapping downstream trimers (Figure 4—figure supplement 1).”
Quote in the paperSubstantial claim - Findingown data
All other mismatches were at least two orders of magnitude slower than their matched counterparts.
“All other mismatches were at least two orders of magnitude slower than their matched counterparts.”
Quote in the paperSubstantial claim - Findingown data
Copying of templates containing two consecutive A or U monomers is made possible by the use of activated helper oligonucleotides.
“The copying of templates containing two consecutive A or U monomers is also made possible by the use of activated helper oligonucleotides (Figure 5).”
Quote in the paperSubstantial claim - Findingown data
Short, activated oligonucleotides are efficient catalysts of high fidelity primer extension with all four RNA monomers.
“Short, activated oligonucleotides – themselves plausibly generated by either templated or untemplated monomer polymerization – are efficient catalysts of high fidelity primer extension with all four RNA monomers.”
Quote in the paperSubstantial claim - Findingcitation
The copying of long mixed template sequences under our conditions compares favorably, both in terms of rate and efficiency, with reactions in which arbitrary RNA templates are copied by highly evolved ribozyme polymerases.
“However, the copying of long mixed template sequences under our conditions compares favorably, both in terms of rate and efficiency with reactions in which arbitrary RNA templates are copied by highly evolved ribozyme polymerases (Wochner et al., 2011) (Figure 6—figure supplement 1).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Wochner et al., 2011 source
Not checked in the free run.
- Findingown data
In bead-assisted sequential primer extension synthesizing HH2 without 2-thiouridine monomers, a high conversion rate was observed at each step (75-95%).
“Despite the fact that no 2-thiouridine monomers were used (in order to protect the catalytic capacity of the ribozyme) a high conversion rate was observed at each step (75–95%) (Figure 6b).”
Quote in the paperSubstantial claim - Findingown data
The fastest mismatch reaction was primer extension by G during copying of a template U, which was approximately 5% as fast as primer extension with A on the same template.
“The fastest mismatch reaction was primer extension by G during copying of a template U, which was approximately 5% as fast as primer extension with A on the same template (Figure 3a, Figure 3—figure supplement 1).”
Quote in the paperSubstantial claim - Findingnone given
Generally, G and C additions were much faster than A or U additions in bead-assisted primer extension.
“Generally, G and C additions were much faster than A or U additions; the reason for the discrepancy between the rates of these reactions on beads and those in solution is unknown.”
Quote in the paperNot a substantial claim - Findingown data
In each case, the rate of primer extension increased by at least two orders of magnitude compared to the corresponding reactions without any helper oligonucleotides.
“In each case, the rate of primer extension increased by at least two orders of magnitude compared to the corresponding reactions without any helper oligonucleotides (Figure 2b).”
Quote in the paperSubstantial claim - Findingown data
A downstream 2-methylimidazole-activated AGC trimer catalyzes template-directed RNA primer extension with all four individual 2-methylimidazole activated monomers - A, G, C and U.
“We then tested the ability of a downstream 2-methylimidazole-activated AGC trimer to catalyze template-directed RNA primer extension with all four individual 2-methylimidazole activated monomers – A, G, C and U.”
Quote in the paperSubstantial claim - Findingown data
The overall fidelity of trimer-assisted polymerization is 98%.
“By averaging the fidelities of the four templates we found the overall fidelity of trimer-assisted polymerization to be 98%.”
Quote in the paperSubstantial claim - Findingown data
Full-length HH2 comprised only 6% of the reaction products in the crude bead-assisted synthesis.
“with full-length HH2 comprising only 6% of the reaction products”
- Also stated in: § Results · plain
“Final yield of the +12 product was 6%.”
Quote in the paperSubstantial claim - Also stated in: § Results · plain
- Findingown data
Our method can only produce sequences >7 nucleotides in one reaction in low yields due to the cross-inhibitory effect of trimers with overlapping binding sites.
“While our method can only produce sequences >7 nucleotides in one reaction in low yields (Figure 6—figure supplement 1) due to the cross-inhibitory effect of trimers with overlapping binding sites (Figure 4—figure supplement 1)”
Quote in the paperSubstantial claim - Premisecitation
Extensive optimization of reaction conditions improved the rate and regiospecificity of polymerization for G and C but not A and U.
“Extensive optimization of reaction conditions with the aim of improving the rate of primer extension with all four monomers, including varying the choice of divalent cation and leaving group, improved the rate and regiospecificity of polymerization for G and C but not A and U (Wu and Orgel, 1992b, c; Deck et al., 2011; Inoue and Orgel, 1981; Hagenbuch et al., 2005; Lohrmann et al., 1980).”
Quote in the paperSubstantial claim0 of 3 sources checked - Premisecitation
The slow rate of primer extension with adenosine and uridine monomers is the most notable reason nonenzymatic copying of all-four-nucleotide sequences has not been possible.
“However, the nonenzymatic copying of sequences containing all four nucleotides has not yet been possible for a number of reasons (Szostak, 2012), most notably the slow rate of primer extension with adenosine and uridine monomers (Joyce et al., 1987; Wu and Orgel, 1992b; Deck et al., 2011).”
Quote in the paperSubstantial claim0 of 3 sources checked - Premisecitation
Two adjacent A or U residues in the template will stop polymerization entirely under normal conditions.
“Indeed, two adjacent A or U residues in the template will stop polymerization entirely (Wu and Orgel, 1992b) under normal conditions; even partial copying requires extreme conditions, such as the eutectic phase of frozen samples (Vogel and Richert, 2007), which are not compatible with replication within protocells.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Wu and Orgel, 1992b
Not checked in the free run.
- Premisecitation
Efficient primer extension requires the presence of two activated monomers adjacent to the primer.
“Furthermore, Orgel et al. showed over 20 years ago that efficient primer extension requires the presence of two activated monomers adjacent to the primer.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Wu and Orgel, 1992a
Not checked in the free run.
- Premisecitation
The reaction is fastest when both monomers are activated with 2-methylimidazole instead of either or both being activated with imidazole.
“Remarkably, the reaction is fastest when both monomers are activated with 2-methylimidazole instead of either or both being activated with imidazole (Wu and Orgel, 1992a), suggesting a possible catalytic role for a physical interaction of the leaving groups of adjacent monomers.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Wu and Orgel, 1992a
Not checked in the free run.
- Interpretationreasoning
The fidelity of primer extension products resulting from the sequential addition of activated monomers, when catalyzed by activated oligomers, is sufficient to sustain a genome long enough to encode active ribozymes.
“The fidelity of the primer extension products resulting from the sequential addition of activated monomers, when catalyzed by activated oligomers, is sufficient to sustain a genome long enough to encode active ribozymes.”
- Also stated in: § Results · plain
“Assuming an error threshold of one mutation per genome (Eigen, 1971), this value of fidelity allows for effective genome sizes of 50 nucleotides, long enough to produce functional ribozymes (Ferré-D'Amaré and Scott, 2010).”
Quote in the paperSubstantial claim0 of 2 sources checked- cites Eigen, 1971 source
Not checked in the free run.
- cites Ferré-D'Amaré and Scott, 2010 source
Not checked in the free run.
- The authors state this as an assumption: An error threshold of one mutation per genome is assumed.
- Also stated in: § Results · plain
- Premisecitation
Nonenzymatic copying of sequences containing all four nucleotides has not yet been possible.
“However, the nonenzymatic copying of sequences containing all four nucleotides has not yet been possible for a number of reasons (Szostak, 2012), most notably the slow rate of primer extension with adenosine and uridine monomers (Joyce et al., 1987; Wu and Orgel, 1992b; Deck et al., 2011).”
Quote in the paperSubstantial claim0 of 3 sources checked - Interpretationreasoning
The slow rate of activated oligonucleotide ligation is explained by the lack of a leaving group one base downstream of the site of the primer extension reaction.
“The slow rate of activated oligonucleotide ligation is also explained by the lack of a leaving group one base downstream of the site of the primer extension reaction: in this case the downstream position corresponds to the second monomer in the oligonucleotide which necessarily has no leaving group as it is linked to the upstream monomer by a phosphodiester linkage.”
Quote in the paperSubstantial claim - Interpretationreasoning
Assuming an error threshold of one mutation per genome, a fidelity of 98% allows for effective genome sizes of 50 nucleotides, long enough to produce functional ribozymes.
“Assuming an error threshold of one mutation per genome (Eigen, 1971), this value of fidelity allows for effective genome sizes of 50 nucleotides, long enough to produce functional ribozymes (Ferré-D'Amaré and Scott, 2010).”
Quote in the paperSubstantial claim0 of 2 sources checked- cites Eigen, 1971 source
Not checked in the free run.
- cites Ferré-D'Amaré and Scott, 2010 source
Not checked in the free run.
- The authors state this as an assumption: An error threshold of one mutation per genome is assumed.
- Interpretationown data
A significant portion of an active ribozyme was synthesized nonenzymatically.
“thus demonstrating that a significant portion of an active ribozyme was synthesized nonenzymatically.”
Quote in the paperSubstantial claim - Interpretationown data
The experiments demonstrate for the first time a simple and robust means of nonenzymatically copying mixed sequence RNA templates.
“The experiments described above demonstrate for the first time a simple and robust means of nonenzymatically copying mixed sequence RNA templates.”
Quote in the paperSubstantial claim - Interpretationcitation
The fastest mismatch is likely due to the formation of a G:U wobble base pair.
“This is likely due to the formation of a G:U wobble base pair (Heuberger et al., 2015), and is consistent with previous studies of nonenzymatic primer extension reactions (Rajamani et al., 2010).”
Quote in the paperNot a substantial claim0 of 2 sources checked - Interpretationreasoning
Template copying in a complex milieu containing both activated monomers and oligomers could occur via a hybrid process combining primer extension with monomers and oligonucleotide ligation.
“Template copying in a complex but realistic milieu containing both activated monomers and oligomers could therefore occur via a hybrid process combining primer extension with monomers and oligonucleotide ligation.”
Quote in the paperSubstantial claim- Rests on an assumption: A prebiotic milieu containing both activated monomers and oligomers is realistic.
- Interpretationreasoning
The disparity in rates between reactions catalyzed by activated vs. unactivated trimers indicates a key role for the leaving group downstream of the polymerizing monomer.
“The disparity in rates between the reactions catalyzed by activated vs. unactivated trimers indicates a key role for the leaving group downstream of the polymerizing monomer.”
Quote in the paperSubstantial claim
- May combine two claims. 11
- The quote only partly states this claim. 1
- Classified as probably not a substantial claim. 7
May combine two claims.
Findingown dataPrimer extension by addition of a single monomer, in the absence of any additional downstream mono- or oligo-nucleotides, was very slow and comparable in rate to the ligation of the primer to a 5′-activated GAG trinucleotide.
“Primer extension by addition of a single monomer, in the absence of any additional downstream mono- or oligo-nucleotides, was very slow and comparable in rate to the ligation of the primer to a 5′-activated GAG trinucleotide (Figure 1c–f, reactions 2 and 3).”
Quote in the paperSubstantial claimThe quote only partly states this claim.
Findingown dataThe primer extension reaction was almost 100 times faster than ligation for a variety of trimer sequences.
“This was true for a variety of trimer sequences (Figure 1—figure supplement 2).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataAdding just the first helper trimer resulted in rapid addition of the first monomer (C) to the primer, followed by slow addition of the first trimer (AGC) to the growing primer.
“Adding just the first helper trimer resulted in rapid addition of the first monomer (C) to the primer, followed by slow addition of the first trimer (AGC) to the growing primer.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn the absence of activated trimers, almost no primer extension was observed; even though all four monomers were present, their combined rate of addition was not measurable.
“In the absence of activated trimers, we observed almost no primer extension (lane 1 Figure 4b); even though all four monomers were present, their combined rate of addition was not measurable.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe reaction durations required to achieve these conversion rates varied widely; the seventh monomer addition required 21 hr to reach 95% while the eighth addition reached the same level of conversion after just 30 min.
“The reaction durations required to achieve these conversion rates varied widely, for example, the seventh monomer addition required 21 hr to reach 95% while the eighth addition reached the same level of conversion after just 30 min.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe crude product mixture was able to function as a catalyst, with 50% ribozyme cleavage yield.
“the product mixture was able to function as a catalyst, with 50% ribozyme cleavage yield”
- Also stated in: § Results · plain
“The third lane shows the extent of the nuclease reaction, 50%, after 16 hr at room temperature.”
Quote in the paperSubstantial claim- Also stated in: § Results · plain
May combine two claims.
Findingown dataAdding two, three or four trimers together resulted in the generation of primers extended by two, three or four monomers respectively.
“Similarly, adding two, three or four trimers together resulted in the generation of primers extended by two, three or four monomers respectively (Figure 4b).”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationDownstream helper oligonucleotides improve the binding of A and U monomers, but the underlying difference in rates remains.
“Moreover, Deck et al. have shown that downstream helper oligonucleotides (Deck et al., 2011) that provide incoming monomers with an additional binding surface improve the binding of A and U monomers, but the underlying difference in rates remains.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Deck et al., 2011 source
Not checked in the free run.
Classified as probably not a substantial claim.
Premisenone givenNonenzymatic copying of RNA templates by suitably activated nucleotides must occur quickly enough to replicate functional RNA sequences faster than they degrade.
“In order to serve as a bridge from prebiotic chemistry to the RNA world (Patel et al., 2015; Powner et al., 2009), the nonenzymatic copying of RNA templates by suitably activated nucleotides (Figure 1a) must occur quickly enough to replicate functional RNA sequences faster than they degrade.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationcitationA physical interaction of the leaving groups of adjacent monomers may play a catalytic role.
“Remarkably, the reaction is fastest when both monomers are activated with 2-methylimidazole instead of either or both being activated with imidazole (Wu and Orgel, 1992a), suggesting a possible catalytic role for a physical interaction of the leaving groups of adjacent monomers.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Wu and Orgel, 1992a
Not checked in the free run.
May combine two claims.
PremisecitationUse of helper oligonucleotides allowed primer extension on a template containing all four bases over several weeks, where previously such templates could not be copied at all.
“The use of helper oligonucleotides allowed primer extension to proceed on a template containing all four bases over the course of several weeks, where previously such templates could not be copied at all.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Deck et al., 2011 source
Not checked in the free run.
May combine two claims.
PremisecitationEven partial copying of templates with adjacent A or U requires extreme conditions, such as the eutectic phase of frozen samples, which are not compatible with replication within protocells.
“Indeed, two adjacent A or U residues in the template will stop polymerization entirely (Wu and Orgel, 1992b) under normal conditions; even partial copying requires extreme conditions, such as the eutectic phase of frozen samples (Vogel and Richert, 2007), which are not compatible with replication within protocells.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Vogel and Richert, 2007 source
Not checked in the free run.
Probably not a substantial claim: may not pass S1 (asserted).
InterpretationreasoningA ribozyme could be synthesized by this approach if monomers were added, along with their attendant trimers, one at a time.
“we reasoned that a ribozyme could be synthesized by this approach if monomers were added, along with their attendant trimers, one at a time (Figure 6).”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
PremisecitationIf nonenzymatic RNA polymerization preceded the RNA world, then it would have been necessary to synthesize RNA sequences long enough to function as ribozymes.
“If nonenzymatic RNA polymerization did indeed precede the RNA world, then it would have been necessary to synthesize RNA sequences long enough to function as ribozymes (Deck et al., 2011).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Deck et al., 2011 source
Not checked in the free run.
May combine two claims.
InterpretationreasoningShort activated oligonucleotides can play dual roles, acting either as catalysts of chain growth by monomer addition, or directly as replication intermediates, since they can also be incorporated into a growing chain by ligation.
“Thus short activated oligonucleotides can play dual roles, acting either as catalysts of chain growth by monomer addition, or directly as replication intermediates, since they can also be incorporated into a growing chain by ligation.”
Quote in the paperSubstantial claim- Rests on an assumption: Short activated oligonucleotides can be generated prebiotically by templated or untemplated monomer polymerization.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll experiments were performed in triplicate or greater.
“Error bars indicate S.E.M; all experiments were performed in triplicate or greater.”
- Also stated in: § Results · plain
“Error bars indicate S.E.M; all experiments were performed in triplicate or greater.”
- Also stated in: § Results · plain
“Error bars indicate S.E.M. All experiments were performed in triplicate or greater.”
Quote in the paperNot a substantial claim- Also stated in: § Results · plain
Probably not a substantial claim: may not pass S3 (load-bearing).
Interpretationown dataThe difference between the fast rates observed in the trimer-assisted addition of a single base and the slower rate of trimer-assisted polymerization of all four bases simultaneously may be due to the competition of trimers for overlapping binding sites on the template.
“The difference between the fast rates observed in the trimer-assisted addition of a single base (Figure 2b) and the slower rate of trimer-assisted polymerization of all four bases simultaneously (Figure 4b) may be due to the competition of trimers for overlapping binding sites on the template.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Premisecitation2-thiouridine monomer is superior to uridine in nonenzymatic primer extension in terms of both increased rate and improved fidelity.
“We also tested the 2-thiouridine monomer, which we have previously shown to be superior to uridine in nonenzymatic primer extension in terms of both increased rate and improved fidelity (Heuberger et al., 2015).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Heuberger et al., 2015 source
Not checked in the free run.
The authors state this as an assumption.
2-methylimidazole-activated nucleotides are suitable models for nonenzymatic template copying despite uncertain prebiotic plausibility.
“Nucleotides activated with 2-methylimidzole on the 5′-phosphate have been used extensively to model nonenzymatic template copying (Deck et al., 2011; Wu and Orgel, 1992c), irrespective of their prebiotic plausibility, and were used in all experiments reported here.”
The authors state this as an assumption.
An error threshold of one mutation per genome is assumed.
“Assuming an error threshold of one mutation per genome (Eigen, 1971), this value of fidelity allows for effective genome sizes of 50 nucleotides, long enough to produce functional ribozymes (Ferré-D'Amaré and Scott, 2010).”
The paper relies on this without support.
Short activated oligonucleotides can be generated prebiotically by templated or untemplated monomer polymerization.
“Short, activated oligonucleotides – themselves plausibly generated by either templated or untemplated monomer polymerization – are efficient catalysts of high fidelity primer extension with all four RNA monomers.”
The paper relies on this without support.
A prebiotic milieu containing both activated monomers and oligomers is realistic.
“Template copying in a complex but realistic milieu containing both activated monomers and oligomers could therefore occur via a hybrid process combining primer extension with monomers and oligonucleotide ligation.”
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 catalytic effect of a downstream, activated nucleotide is much stronger for an oligonucleotide than a mononucleotide at the downstream position.
“this effect is much stronger for an oligonucleotide than a mononucleotide at the downstream position”
From Protocells and RNA Self-Replication (2018) · cites this paper as Prywes et al. 2016
The rate of ligation of a primer to a 5'-imidazole-activated oligonucleotide is very slow compared with the rate of primer extension with similarly activated monomers.
“the rate of ligation of a primer to a 5 ′ -imidazole-activated oligonucleotide is very slow compared with the rate of primer extension with similarly activated monomers”
From Protocells and RNA Self-Replication (2018) · cites this paper as Prywes et al. 2016
During template-directed non-enzymatic RNA polymerization in solution using activated nucleotide monomers, hydrolyzed, deactivated monomers accumulate over time, and these hinder template-directed polymerization by competitively occupying the complementary sites on the template strand.
“As a hypothetical example, during template-directed non-enzymatic RNA polymerization in solution using activated nucleotide monomers [72,73], hydrolyzed, deactivated monomers accumulate over time, and these hinder template-directed polymerization by competitively occupying the complementary sites on the template strand [72].”
From Polyesters as a Model System for Building Primitive Biologies from Non-Biological Prebiotic Chemistry (2020) · cites this paper as [72]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Figure 4 reaction conditions conflict with the methods
MajorThe Figure 4 caption and the methods give different primer, template, buffer, and monomer concentrations for the multiple-monomer reaction. They also specify different trimer concentrations.
“Reaction conditions: 2.5 μM primer, 2.5 μM template, 100 mM HEPES pH 8.0, 100 mM MgCl2, 20 mM monomer, 100 μM trimer.”
“Primer extension reactions exploring the addition of multiple monomers and trimers in one reaction had the following conditions: 100 mM MgCl2, 200 mM Tris-HCl pH 8, 25 mM monomers, 500 μM 2-methylimidazole-activated AGC, CGG and GGG, 100 μM activated GCG, 10 μM primer and 11 μM template.”
Figure 2 monomer concentration differs between caption and methods
MajorThe Figure 2 caption specifies 50 mM monomer, while the methods describe the corresponding reactions using 25 mM monomer.
“Reaction conditions: 10 μM primer, 11 μM template, 200 mM Tris pH 8.0, 100 mM MgCl2, 50 mM monomer, 10 mM 2-MeImpAG, 1 mM 2-MeImpAGC, 1 mM 2-MeImpAGGC.”
“Primer extension reactions exploring the effect of different monomers and different oligomers on the rate of polymerization were conducted under the following conditions: 100 mM MgCl2, 200 mM Tris-HCl pH 8, 25 mM monomer, 1 mM trimer, 10 μM primer and 11 μM template.”
Figure 2 activated AG concentration differs between caption and methods
MajorThe Figure 2 caption specifies 10 mM 2-MeImpAG, but the methods specify 1 mM activated AG for the helper-oligomer experiments.
“10 mM 2-MeImpAG, 1 mM 2-MeImpAGC, 1 mM 2-MeImpAGGC.”
“The downstream helper oligomers were added in the following concentrations: 25 mM 2-MeImpA and 1 mM activated oligonuleotide (AG, AGC or AGGC).”
Hammerhead cleavage assay duration differs
MinorThe results and Figure 6 caption report cleavage after 16 hours, whereas the assay methods specify a 20-hour incubation.
“The third lane shows the extent of the nuclease reaction, 50%, after 16 hr at room temperature.”
“Finally, the reaction was incubated at room temperature with 200 mM MgCl2, 200 mM Tris-HCl pH 8 and 250 nM Cy5 labeled HH1 RNA (Cy5 - 5′ - CGC GCC GAA ACA CCG UGU CCC AGU C - 3′) for 20 hr.”
DNA-mediated release time differs
MinorThe Figure 6 caption says the product was released at 95°C over 2 minutes, while the cleavage-assay methods specify a 1-minute incubation at that temperature.
“The product of the last bead-assisted reaction was removed from the bead by the addition of the an excess of the DNA sequence of the extended primer at 95°C over 2 min.”
“174 μM DNA complement was added in 0.01% (v/v) Tween 20 and the beads were incubated at 95°C for 1 min.”
The citation lists haven’t been fetched yet
References 30 · Cited by 141
- Authors
- Prywes, N., Blain, J. C., Del Frate, F., & Szostak, J. W.
- Affiliations
- Howard Hughes Medical Institute (US)
- Department of Molecular Biology and Center for Computational and Integrative Biology, Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, United States
- Corresponding author
- Szostak, Jack W
- Venue
- eLife
- Publisher
- eLife Sciences Publications, Ltd
- Published
- 2016-06-28
- Volume
- 5
- ISSN
- 2050-084X (ISSN-L)
- PMC
- PMC4959843
- OpenAlex
- W2467190354
- Semantic Scholar
- e9e2609fd8163f266196f68913e58681d1b5e805
- Cited by
- 141
- References
- 30
- Citations by year
- 2026: 8 · 2025: 13 · 2024: 16 · 2023: 9 · 2022: 14 · 2021: 11 · 2020: 17 · 2019: 18 · 2018: 20 · 2017: 13 · 2016: 2
- FWCI
- 8.60
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- eLife (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Simons Foundation (290363)
- National Science Foundation (DGE1144152)
- 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 › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- primer extension, RNA world, prebiotic chemistry, hammerhead ribozyme, protocells, nucleotide synthesis
- MeSH terms
- Oligonucleotides, RNA, RNA, Catalytic, Polymerization
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