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Enzymatic primer-extension with glycerol-nucleoside triphosphates on DNA templates.

Chen JJ, Tsai CH, Cai X, Horhota AT, McLaughlin LW, Szostak JW - PLoS ONE (2009)

Bottom Line: Steady-state kinetic experiments suggested that GNA synthesis by Therminator was affected by both decreased catalytic rates and weakened substrate binding, especially for pyrimidines.This led to more efficient incorporation of gC, but not gT.We suggest that directed evolution of Therminator might lead to mutants with improved substrate binding and catalytic efficiency.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.

ABSTRACT

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.

Show MeSH
Structures of GNA, TNA, FNA, and DNA (RNA).The backbone similarity is highlighted in red.
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pone-0004949-g001: Structures of GNA, TNA, FNA, and DNA (RNA).The backbone similarity is highlighted in red.

Mentions: The ribose and 2-deoxyribose components of the backbones of RNA and DNA have long been thought to play a critical role in stabilizing Watson-Crick base pairing and maintaining fidelity in polymerase-mediated information transfer. Having survived billions of years of evolution on Earth, ribose has been considered to be one of the indispensable building blocks of contemporary life [1]. However, recent studies from the Eschenmoser group of nucleic acid analogs with altered sugar-phosphate backbones demonstrate dramatically that D-ribofuranose is not a chemical requirement for the ability of a prebiotically plausible nucleic acid to function as the genetic information carrier [2]. Indeed, a nucleic acid with a simpler backbone structure, such as (3′→2′)-α-L-threose nucleic acid (TNA, Figure 1), might have been an evolutionary progenitor of DNA or RNA [3].


Enzymatic primer-extension with glycerol-nucleoside triphosphates on DNA templates.

Chen JJ, Tsai CH, Cai X, Horhota AT, McLaughlin LW, Szostak JW - PLoS ONE (2009)

Structures of GNA, TNA, FNA, and DNA (RNA).The backbone similarity is highlighted in red.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654545&req=5

pone-0004949-g001: Structures of GNA, TNA, FNA, and DNA (RNA).The backbone similarity is highlighted in red.
Mentions: The ribose and 2-deoxyribose components of the backbones of RNA and DNA have long been thought to play a critical role in stabilizing Watson-Crick base pairing and maintaining fidelity in polymerase-mediated information transfer. Having survived billions of years of evolution on Earth, ribose has been considered to be one of the indispensable building blocks of contemporary life [1]. However, recent studies from the Eschenmoser group of nucleic acid analogs with altered sugar-phosphate backbones demonstrate dramatically that D-ribofuranose is not a chemical requirement for the ability of a prebiotically plausible nucleic acid to function as the genetic information carrier [2]. Indeed, a nucleic acid with a simpler backbone structure, such as (3′→2′)-α-L-threose nucleic acid (TNA, Figure 1), might have been an evolutionary progenitor of DNA or RNA [3].

Bottom Line: Steady-state kinetic experiments suggested that GNA synthesis by Therminator was affected by both decreased catalytic rates and weakened substrate binding, especially for pyrimidines.This led to more efficient incorporation of gC, but not gT.We suggest that directed evolution of Therminator might lead to mutants with improved substrate binding and catalytic efficiency.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.

ABSTRACT

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.

Show MeSH