Enzymatic Primer-Extension with Glycerol-Nucleoside Triphosphates on DNA Templates
Open accessAbstract
BACKGROUND: Glycerol nucleic acid (GNA) has an acyclic phosphoglycerol backbone repeat-unit, but forms stable duplexes based on Watson-Crick base-pairing. Because of its structural simplicity, GNA is of particular interest with respect to the possibility of evolving functional polymers by in vitro selection. Template-dependent GNA synthesis is essential to any GNA-based selection system. PRINCIPAL FINDINGS: In this study, we investigated the ability of various DNA polymerases to use glycerol-nucleoside triphosphates (gNTPs) as substrates for GNA synthesis on DNA templates. Therminator DNA polymerase catalyzes quantitative primer-extension by the incorporation of two glyceronucleotides, with much less efficient extension up to five glyceronucleotides. Steady-state kinetic experiments suggested that GNA synthesis by Therminator was affected by both decreased catalytic rates and weakened substrate binding, especially for pyrimidines. In an attempt to improve pyrimidine incorporation by providing additional stacking interactions, we synthesized two new gNTP analogs with 5-propynyl substituted pyrimidine nucleobases. This led to more efficient incorporation of gC, but not gT. CONCLUSIONS: We suggest that directed evolution of Therminator might lead to mutants with improved substrate binding and catalytic efficiency.
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A world of GNA is possible.
“It has been shown that the corresponding glycolic nucleic acid can form helical molecules, opening the possibility of a world of GNA (Chen et al. 2009;Johnson et al. 2011).”
From The Emergence of Life (2019) · cites this paper as Chen et al. 2009
Glycolic nucleic acid can form helical molecules.
“It has been shown that the corresponding glycolic nucleic acid can form helical molecules, opening the possibility of a world of GNA (Chen et al. 2009;Johnson et al. 2011).”
From The Emergence of Life (2019) · cites this paper as Chen et al. 2009
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References 31 · Cited by 29
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- Authors
- Chen, J. J., Tsai, C.-H., Cai, X., Horhota, A. T., McLaughlin, L. W., & Szostak, J. W.
- Affiliations
- Howard Hughes Medical Institute (US)
- Boston College (US)
- Corresponding author
- Szostak, Jack W.
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2009-03-23
- Volume
- 4
- Issue
- 3
- Pages
- e4949
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC2654545
- OpenAlex
- W2051573208
- Cited by
- 29
- References
- 31
- Citations by year
- 2025: 2 · 2023: 4 · 2022: 2 · 2021: 2 · 2020: 1 · 2019: 5 · 2017: 2 · 2016: 1 · 2015: 2 · 2013: 1 · 2012: 2
- FWCI
- 0.94
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Science Foundation (0434507, 0451488)
- Howard Hughes Medical Institute
- Division of Chemistry
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
- Keywords
- primer extension, polymerase kinetics, substrate binding, in vitro selection, directed evolution
- MeSH terms
- DNA, DNA-Directed DNA Polymerase, Glycerol, Nucleic Acid Conformation, Templates, Genetic, Molecular Structure, DNA Primers
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