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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 glyceronucleoside triphosphates (gNTPs).
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pone-0004949-g002: Structures of glyceronucleoside triphosphates (gNTPs).

Mentions: In addition to exploring GNA molecules as a genetic information carrier, we are also interested in probing their ligand binding and catalytic abilities. We envision that an in vitro selection strategy, similar to that used in RNA aptamer or artificial ribozyme selection [8], can be used to isolate functional GNA constructs. This selection strategy requires a polymerase that can synthesize GNA in a template-directed fashion. We have previously synthesized the four canonical glycerol-nucleoside triphosphates (gNTPs with A, G, T, or C, Figure 2) and demonstrated that several DNA polymerases can incorporate a single glyceronucleotide onto a DNA primer/template using gNTPs as the substrates [9]. Incorporation of a second glyceronucleotide was not observed in that study, which we attributed to the poorly constrained nature of the acyclic glycerophosphate backbone of GNA and/or the unstable nature of a GNA/DNA duplex [9].


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 glyceronucleoside triphosphates (gNTPs).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004949-g002: Structures of glyceronucleoside triphosphates (gNTPs).
Mentions: In addition to exploring GNA molecules as a genetic information carrier, we are also interested in probing their ligand binding and catalytic abilities. We envision that an in vitro selection strategy, similar to that used in RNA aptamer or artificial ribozyme selection [8], can be used to isolate functional GNA constructs. This selection strategy requires a polymerase that can synthesize GNA in a template-directed fashion. We have previously synthesized the four canonical glycerol-nucleoside triphosphates (gNTPs with A, G, T, or C, Figure 2) and demonstrated that several DNA polymerases can incorporate a single glyceronucleotide onto a DNA primer/template using gNTPs as the substrates [9]. Incorporation of a second glyceronucleotide was not observed in that study, which we attributed to the poorly constrained nature of the acyclic glycerophosphate backbone of GNA and/or the unstable nature of a GNA/DNA duplex [9].

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