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Cited by 78Origins and Evolution of LifeRNA and protein synthesis mechanismsDNA and Nucleic Acid Chemistry

Replacing Uridine with 2-Thiouridine Enhances the Rate and Fidelity of Nonenzymatic RNA Primer Extension

Open access
Heuberger, Benjamin D., Pal, Ayan, Del Frate, Francesca, Topkar, Ved V., Szostak, Jack W.·2015·Journal of the American Chemical Society
DOI 10.1021/jacs.5b00445PMC PMC4985000
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Abstract

The nonenzymatic replication of RNA oligonucleotides is thought to have played a key role in the origin of life prior to the evolution of ribozyme-catalyzed RNA replication. Although the copying of oligo-C templates by 2-methylimidazole-activated G monomers can be quite efficient, the copying of mixed sequence templates, especially those containing A and U, is particularly slow and error-prone. The greater thermodynamic stability of the 2-thio-U(s2U):A base pair, relative to the canonical U:A base pair, suggests that replacing U with s2U might enhance the rate and fidelity of the nonenzymatic copying of RNA templates. Here we report that this single atom substitution in the activated monomer improves both the kinetics and the fidelity of nonenzymatic primer extension on mixed-sequence RNA templates. In addition, the mean lengths of primer extension products obtained with s2U is greater than those obtained with U, augmenting the potential for nonenzymatic replication of heritable function-rich sequences. We suggest that noncanonical nucleotides such as s2U may have played a role during the infancy of the RNA world by facilitating the nonenzymatic replication of genomic RNA oligonucleotides.

Integrity signals

Registry

Crossref · 10.1021/jacs.5b00445

Cites

31 of 31 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 25 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Primer extension following a G:U mismatch was highly error prone, with an error rate of almost 40%.

    “Remarkably, primer extension following a G:U mismatch was highly error prone, with an error rate of almost 40%.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Next-generation sequencing accurately measures the fidelity of nonenzymatic primer extension products.
  • Findingown data

    With the 5′-C5A template, incorporation of 2-MeImpU, 2-MeImps2U, or 2-MeImps2T in the presence of 2-MeImpG led to kmax values of 0.26 ± 0.063, 0.96 ± 0.11, and 4.4 ± 0.45 h-1, respectively.

    “The incorporation of either 2-MeImpU, 2-MeImps2U or 2-MeImps2T in the presence of 2-MeImpG led to kmax values of 0.26 ± 0.063, 0.96 ± 0.11, and 4.4 ± 0.45 h–1, respectively”
    Quote in the paperSubstantial claim
  • Findingown data

    The magnitude of the stalling effect is ca. 20-fold.

    “The magnitude of this effect (ca. 20-fold, Figure 5) is surprising and should be examined in additional sequence contexts.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Next-generation sequencing accurately measures the fidelity of nonenzymatic primer extension products.
  • Findingnone given

    Under different template and buffer conditions, the rate of primer extension consistently followed the order s2T > s2U > U.

    “Under different template and buffer conditions, the rate of primer extension consistently followed the order s2T > s2U > U.”
    Quote in the paperSubstantial claim
  • Findingown data

    Thiolation of U or T in the template had little effect on the rate of primer extension.

    “However, as with the homopolymer templates, thiolation of U or T in the template had little effect on the rate of primer extension.”
    Quote in the paperNot a substantial claim
  • May combine two claims. 13
  • Classified as probably not a substantial claim. 11

  • May combine two claims.

    Findingown data

    The enhanced rate and fidelity of template copying was only observed with 2-thio-U or -T as the activated monomer, and no significant effect was observed when these modified nucleotides were present in the template strand.

    “Surprisingly, this enhanced rate and fidelity of template copying was only observed with 2-thio-U or -T as the activated monomer, and no significant effect was observed when these modified nucleotides were present in the template strand.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    G:U mispairing at the second position was diminished from 4.3% with 2-MeImpU to 1.6% with 2-MeImps2U and 2.0% with 2-MeImps2T, corresponding to a 2-3 fold improvement in fidelity.

    “We found that G:U mispairing at the second position was diminished from 4.3% in the case of 2-MeImpU to 1.6% with 2-MeImps2U and 2.0% with 2-MeImps2T (Figure 5a; see also Supporting Information Table 2), corresponding to a 2–3 fold improvement in fidelity with the 2-thiolated U or T monomers.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Next-generation sequencing accurately measures the fidelity of nonenzymatic primer extension products.

  • May combine two claims.

    Findingown data

    Replacing U with s2U in the activated monomer improves both the kinetics and fidelity of nonenzymatic primer extension on mixed-sequence RNA templates.

    “Here we report that this single atom substitution in the activated monomer improves both the kinetics and the fidelity of nonenzymatic primer extension on mixed-sequence RNA templates.”
    • Also stated in: § Discussion · plain
      “A single-atom substitution of oxygen by sulfur at the 2-position of the activated uridine nucleotide 2-MeImpU significantly improves the rate and fidelity of nonenzymatic template-directed primer extension.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Next-generation sequencing accurately measures the fidelity of nonenzymatic primer extension products.

  • May combine two claims.

    Findingown data

    There was a 5-6 fold drop in the amount of primer-UCC when 2-thiolated U-derivatives were used, with only 4.3% and 2.9% C incorporation at position 3 with 2-MeImps2U and 2-MeImps2U, respectively.

    “We noted a 5–6 fold drop in the amount of primer-UCC when 2-thiolated U-derivatives were used, with only 4.3% and 2.9% C incorporation at position 3 when primer extension was carried out with 2-MeImps2U and 2-MeImps2U, respectively”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Next-generation sequencing accurately measures the fidelity of nonenzymatic primer extension products.

  • May combine two claims.

    Findingown data

    Incorporation of 2-MeImpA in the presence of 2-MeImpG was approximately an order of magnitude faster on the single-U, s2U, or s2T templates than on the corresponding homopolymeric templates, with observed rates of 0.83 ± 0.045, 0.38 ± 0.019, and 0.78 ± 0.024 h-1, respectively.

    “we found that the incorporation of 2-MeImpA in the presence of 2-MeImpG was approximately an order of magnitude faster on the single-U, s2U or s2T templates than on the corresponding homopolymeric templates, with observed rates of 0.83 ± 0.045, 0.38 ± 0.019, and 0.78 ± 0.024 h–1, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both G:U and A:C misincorporation largely terminated further primer extension, and the small amount of continued primer extension was highly error prone.

    “we observed that both G:U and A:C misincorporation largely terminated further primer extension and, furthermore, that the small amount of continued primer extension was highly error prone.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Next-generation sequencing accurately measures the fidelity of nonenzymatic primer extension products.

  • May combine two claims.

    Findingown data

    There was insignificant differentiation between U, s2U, or s2T when they were templating primer extension in the context of 5′-C5U*-3′ templates, and the distributions of product length were comparable in all three cases.

    “Interestingly, there was insignificant differentiation between U, s2U, or s2T when they were templating primer extension in this context, and the distributions of product length were comparable in all three cases”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Next-generation sequencing accurately measures the fidelity of nonenzymatic primer extension products.

  • May combine two claims.

    Findingown data

    For the correct 2-nucleotide extension product primer-UC, there were 53,134 sequences and 13,077 sequences corresponding to continued primer extension by at least one more nucleotide, i.e., about 20% continued primer extension.

    “we observed 53 134 sequences corresponding to the correct 2-nucleotide extension product primer-UC, and 13 077 sequences corresponding to continued primer extension by at least one more nucleotide, i.e., about 20% continued primer extension.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Next-generation sequencing accurately measures the fidelity of nonenzymatic primer extension products.

  • May combine two claims.

    Premisecitation

    Replacing U with s2U stabilizes base pairing with A while modestly destabilizing wobble pairing with G.

    “We sought to address these challenges by replacing U with s2U, a nucleobase known to significantly stabilize base pairing with A while modestly destabilizing wobble pairing with G.14,15”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [14] Kumar, 1997, Nucleic Acids Res. source

      Not checked in the free run.

    • cites [15] Testa, 1999, Biochemistry source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationreasoning

    Enhancing the ability of RNA to make large jumps through sequence space may be particularly important in facilitating the emergence of novel RNA-coded functions.

    “Enhancing the ability of RNA to make large jumps through sequence space may be particularly important in facilitating the emergence of novel RNA-coded functions.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Using 2-methylimidazole-activated mononucleotides to copy short C-rich oligonucleotide templates was a successful model system.

    “Their most successful model system involved the use of 2-methylimidazole-activated mononucleotides to nonenzymatically copy short C-rich oligonucleotide templates.4”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Inoue, 1983, Science source

      Not checked in the free run.

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

    Premisecitation

    In RNA many occurrences of s2U also contain substitutions at the 5-position.

    “in RNA many occurrences of s2U also contain substitutions at the 5-position.19,20”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [19] Agris, 2004, Nucleic Acids Res. source

      Not checked in the free run.

    • cites [20] Machnicka, 2013, Nucleic Acids Res. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Nonenzymatic primer extension in another system with 3′-amino-2′,3′-dideoxynucleotides exhibits enhanced rates and fidelity upon replacement of T with s2T.

    “we have recently reported that nonenzymatic primer extension in another system, in which primer, template, and monomers are 3′-amino-2′,3′-dideoxynucleotides, exhibits enhanced rates and fidelity upon replacement of T with s2T.26”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Zhang, 2013, Proc. Natl. Acad. Sci. U. S. A. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Nonenzymatic replication of RNA oligonucleotides is thought to have been important for the origin of life before ribozyme-catalyzed RNA replication evolved.

    “The nonenzymatic replication and transmission of genetic information may have played an important role in the transition from prebiotic chemistry to cellular life by enabling the evolution of selectively advantageous ribozyme catalysts prior to the emergence of ribozyme RNA polymerases.1”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [1] Szostak, 2012, J. Syst. Chem. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Replacing U with s2U or s2T should improve the overall rate of primer extension not only because of the increased rate at which the 2-thio monomers are incorporated but also because the increased accuracy of primer extension will lead to less stalling after mismatches.

    “Nevertheless, we now expect that replacing U with s2U or s2T should improve the overall rate of primer extension not only because of the increased rate at which the 2-thio monomers are incorporated but also because the increased accuracy of primer extension will lead to less stalling after mismatches.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    s2T stabilizes tRNAs from extreme thermophiles, most likely through stabilization of the 3′-endo sugar conformation.

    “s2T stabilizes tRNAs from extreme thermophiles,22,23 most likely through stabilization of the 3′-endo sugar conformation.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [22] Watanabe, 1979, Biochem. Biophys. Res. Commun. source

      Not checked in the free run.

    • cites [23] Davanloo, 1979, Nucleic Acids Res. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Weaker hydrogen bonding between the sulfur of s2U(T) and the imino proton of G would weaken wobble pairing, favoring correct binding of C, and together with a further distorted geometry may account for the decreased formation of U:G mismatches observed with s2U and s2T.

    “Weaker hydrogen bonding between the sulfur of s2U(T) and the imino proton of G would weaken wobble pairing, favoring correct binding of C, and that together with a further distorted geometry may account for the decreased formation of U:G mismatches observed with s2U and s2T.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationcitation

    Such clustered mutations may speed the exploration of sequence space and the optimization of functions under selection.

    “Such clustered mutations may speed the exploration of sequence space and the optimization of functions under selection.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The decreased frequency of A:C mismatches is attributed to the faster reaction rate of s2U or s2T (relative to U) when paired with A; effectively s2U and s2T outcompete C so that A:C mismatches do not have time to form.

    “We attribute the decreased frequency of A:C mismatches to the faster reaction rate of s2U or s2T (relative to U) when paired with A; effectively s2U and s2T outcompete C so that A:C mismatches do not have time to form.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    s2T improves base pair discrimination in the context of PCR.

    “s2T also improves base pair discrimination in the context of PCR.27”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Sismour, 2005, Nucleic Acids Res. source

      Not checked in the free run.

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

    Premisecitation

    Replacing thymine with 2-thiothymine in DNA is stabilizing.

    “Replacing thymine with 2-thiothymine in DNA is also stabilizing,18”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Sintim, 2006, J. Am. Chem. Soc. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For pseudo-first-order rate determinations, primer extension reactions were studied for a maximum of 50 min, much less than the 2-5 day half-time of hydrolysis of the activated nucleotides.

    “For pseudo-first-order rate determinations, primer extension reactions were studied for a maximum of 50 min, much less than the 2–5 day half-time of hydrolysis of the activated nucleotides.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    The larger and more diffuse electron cloud of sulfur vs oxygen makes it more polarizable and may contribute to better stacking.

    “In addition the larger and more diffuse electron cloud of sulfur vs oxygen makes it more polarizable and hence may contribute to better stacking.15,17”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [15] Testa, 1999, Biochemistry source

      Not checked in the free run.

    • cites [17] Saenger, 1984 source

      Not checked in the free run.

23 of 23

  • The paper relies on this without support.

    Potentially functional RNA sequences are more likely to contain all four nucleotides.

    “they are not directly relevant to potentially functional sequences, which are more likely to contain all four nucleotides.”

  • The paper relies on this without support.

    The mixed sequence templates used (C5N pattern) provide a more realistic setting for primer extension than homopolymeric templates.

    “We therefore used a set of mixed sequence templates to examine primer extension in a more realistic setting.”

  • The paper relies on this without support.

    Next-generation sequencing accurately measures the fidelity of nonenzymatic primer extension products.

    “We used next generation sequencing (NGS) to assess the fidelity of the primer extension products obtained on mixed sequence templates, in the presence of competing activated monomers.”
3 of 3

Claims other papers make about this one

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

  • Template copying experiments with 2MeI-activated monomers suggest that 2-thio-U reduces wobble-pairing errors from 4%-5% to about 2% at template G positions, suggesting that an overall error rate of 1%-2% may be possible.

    “Template copying experiments with 2MeI-activated monomers suggest that this substitution reduces wobble-pairing errors from 4%-5% to ∼2% at template G positions, suggesting that an overall error rate of 1%-2% may be possible”

    From Protocells and RNA Self-Replication (2018) · cites this paper as Heuberger et al. 2015

    The Results report that G:U mispairing at the tested G position fell from 4.3% with 2-MeImpU to 1.6% with 2-MeImps2U, supporting the stated reduction. The cited paper does not give an overall copying error rate of 1%–2% or state that this rate may be possible; that is an extrapolation from a position-specific result. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Replacing U with 2-thio-U reduces both of these sources of errors because the 2-thio-U•A base pair is almost as strong as a C•G base pair.

    “replacing U with 2-thio-U reduces both of these sources of errors because the 2-thio-U•A base pair is almost as strong as a C•G base pair.”

    From Protocells and RNA Self-Replication (2018) · cites this paper as Heuberger et al. 2015

    The Results report that replacing U with 2-thio-U reduced both G:U and A:C mispairing. The Introduction says 2-thio-U stabilizes pairing with A, but does not establish that the pair is almost as strong as C:G. The Discussion attributes the two error reductions to different mechanisms, rather than to that strength comparison. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Replacement of U by 2-thio-U also results in faster and more efficient copying of templates that contain A residues.

    “Replacement of U by 2-thio-U also results in faster and more efficient copying of templates that contain A residues.”

    From Protocells and RNA Self-Replication (2018) · cites this paper as Heuberger et al. 2015

  • 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).”

    From Nonenzymatic copying of RNA templates containing all four letters is catalyzed by activated oligonucleotides (2016) · cites this paper as Heuberger et al., 2015

  • 2-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).”

    From Nonenzymatic copying of RNA templates containing all four letters is catalyzed by activated oligonucleotides (2016) · cites this paper as Heuberger et al., 2015

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Authors
Heuberger, B. D., Pal, A., Del Frate, F., Topkar, V. V., & Szostak, J. W.
Affiliations
  • Howard Hughes Medical Institute (US)
  • Massachusetts General Hospital , , , ,
Corresponding author
Szostak, Jack W.
ORCID
Venue
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Published
2015-02-05
Volume
137
Issue
7
Pages
2769-2775
ISSN
  • 0002-7863 (ISSN-L)
  • 1520-5126
DOI
10.1021/jacs.5b00445
PMC
PMC4985000
OpenAlex
W2317913417
Cited by
78
References
31
Citations by year
2026: 4 · 2025: 6 · 2024: 4 · 2023: 2 · 2022: 7 · 2021: 12 · 2020: 7 · 2019: 8 · 2018: 9 · 2017: 9 · 2016: 8 · 2015: 2
FWCI
5.31
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of the American Chemical Society (publishedVersion, other-oa)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Howard Hughes Medical Institute (NA)
  • Division of Chemistry (CHE-0809413)
  • Simons Foundation (NA)
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
2-thiouridine, RNA replication, origin of life, RNA world, replication fidelity
MeSH terms
Kinetics, Models, Molecular, Nucleic Acid Conformation, RNA, Thiouridine

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