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Cited by 7DNA and Nucleic Acid ChemistryAdvanced biosensing and bioanalysis techniquesRNA and protein synthesis mechanisms

High Fidelity Enzyme‐Free Primer Extension with an Ethynylpyridone Thymidine Analog

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Han, Jianyang, Kervio, Eric, Richert, Clemens·2021·Chemistry – A European Journal
DOI 10.1002/chem.202102996PMC PMC9293356
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Abstract

High fidelity base pairing is important for the transmission of genetic information. Weak base pairs can lower fidelity, complicating sequencing, amplification and replication of DNA. Thymidine 5′‐monophosphate (TMP) is the most weakly pairing nucleotide among the canonical deoxynucleotides, causing high errors rates in enzyme‐free primer extension. Here we report the synthesis of an ethynylpyridone C‐nucleoside analog of 3′‐amino‐2′,3′‐dideoxythymidine monophosphate and its incorporation in a growing strand by enzyme‐free primer extension. The ethynylpyridone C‐nucleotide accelerates extension more than five‐fold, reduces misincorporation and readily displaces TMP in competition experiments. The results bode well for the use of the C‐nucleoside as replacements for thymidine in practical applications.

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  • A limitation remains the reaction time, requiring 12-20 h for each cycle with a very large excess of nucleotides and poor reaction profiles for the weakly pairing bases, although modified derivatives were shown to alleviate this limitation.

    “A limitation remains the reaction time, requiring 12–20 h for each cycle with a very large excess of nucleotides and poor reaction profiles for the weakly pairing bases, although modified derivatives were shown to alleviate this limitation.17”

    From Sequencing of <scp>d</scp>/<scp>l</scp>-DNA and XNA by Templated-Synthesis (2025) · cites this paper as [17]

    The primer-extension results (section labeled Abstract; page unavailable) report 20 h per extension step with 40 mM monomers. They also report poor fidelity with thymine and a fivefold acceleration and improved fidelity with an ethynylpyridone analog. Our copy does not establish the claimed 12–20 h range or quantify the monomers as a very large excess over primer. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Richert and co-workers recognized the potential of enzyme-free primer extension for genotyping and sequencing with reversible termination.

    “In addition, Richert and co-workers also recognized the potential of enzyme-free primer extension for genotyping15 and sequencing with reversible termination.16,17”

    From Sequencing of <scp>d</scp>/<scp>l</scp>-DNA and XNA by Templated-Synthesis (2025) · cites this paper as [17]

    The paper describes enzyme-free primer extension with Azoc-based reversible termination and says that fidelity matters for sequencing (both passages labeled Abstract; page unavailable). It does not mention genotyping or explicitly propose sequencing using this reversible-termination method. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

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Cites

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Cited by

Sorted by citations

Authors
Han, J., Kervio, E., & Richert, C.
Affiliations
University of Stuttgart (DE)
Corresponding author
Richert, Clemens
ORCID
Venue
Chemistry – A European Journal
Publisher
Wiley
Published
2021-10-08
Volume
27
Issue
64
Pages
15918-15921
ISSN
  • 0947-6539 (ISSN-L)
  • 1521-3765
DOI
10.1002/chem.202102996
PMC
PMC9293356
OpenAlex
W3199089120
Cited by
7
References
4
Citations by year
2026: 1 · 2025: 1 · 2024: 2 · 2022: 3
FWCI
0.39
Type
journal article
Language
en
Open access
Licence
Locations
  • Chemistry - A European Journal (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • Deutsche Forschungsgemeinschaft (project-ID 364653263 - TRR 235)
  • Volkswagen Foundation (Az 92 768)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
Keywords
thymidine analogs, thymidine 5'-monophosphate, misincorporation, nucleoside synthesis, DNA replication fidelity
MeSH terms
DNA, DNA Replication, Kinetics, Nucleotides, Thymidine, DNA Primers, Base Pairing

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