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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
Synthesis of 5-propynyluridine glyceronucleoside triphosphate (1u).
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pone-0004949-g004: Synthesis of 5-propynyluridine glyceronucleoside triphosphate (1u).

Mentions: In order to test the hypothesis that poor enzymatic incorporation of pyrimidine gNTPs reflects poor base-stacking, we synthesized the C-5-propynyl substituted pyrimidine nucleoside triphosphates (Figure 2). This co-planar nucleobase modification has been proposed to increase base stacking and hydrophobic interactions between base pairs [11]. In the case of GNA synthesis, we thought that a 5-propynyl group in pyrimidine gNTPs might improve binding to the primer/template complex in the active site of polymerase. The synthetic schemes for the preparation of 5-propynyl substituted gNTPs (gUpropTP and gCpropTP, or 1u and 1c) are shown in Figures 4 and 5. Two key intermediates, the 5-propynyl pyrimidine glycerol-nucleosides (4u and 5c), were prepared from 5-iodo-substituted precursors (3u and 3c) and propyne by Sonogashira coupling (Figure 4 and 5) [12], [13]. A di-butylaminomethylidene group was used to protect the exocyclic amine of 3c instead of an acetyl or benzoyl group in order to avoid a potential cyclization side-reaction involving the amide and the 5-propynyl group [14]. The presence of a propynyl group in 4u and 5c was confirmed by the characteristic chemical shift (4–5 ppm) of the methyl carbon in 13C NMR [15] together with 1H-NMR and ESI-MS analysis. gUpropTP and gCpropTP (1u and 1c) were synthesized from the corresponding nucleosides (5u and 6c) using the one-pot, salicylchlorophosphorin approach developed by Ludwig and Eckstein [16]. The final purified products 1u and 1c were characterized by 1H- and 31P-NMR and by ESI-MS. In addition, 1u and 1c have similar UV absorption profiles to those reported for 5-propynyl-deoxyribonucleosides [15].


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)

Synthesis of 5-propynyluridine glyceronucleoside triphosphate (1u).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004949-g004: Synthesis of 5-propynyluridine glyceronucleoside triphosphate (1u).
Mentions: In order to test the hypothesis that poor enzymatic incorporation of pyrimidine gNTPs reflects poor base-stacking, we synthesized the C-5-propynyl substituted pyrimidine nucleoside triphosphates (Figure 2). This co-planar nucleobase modification has been proposed to increase base stacking and hydrophobic interactions between base pairs [11]. In the case of GNA synthesis, we thought that a 5-propynyl group in pyrimidine gNTPs might improve binding to the primer/template complex in the active site of polymerase. The synthetic schemes for the preparation of 5-propynyl substituted gNTPs (gUpropTP and gCpropTP, or 1u and 1c) are shown in Figures 4 and 5. Two key intermediates, the 5-propynyl pyrimidine glycerol-nucleosides (4u and 5c), were prepared from 5-iodo-substituted precursors (3u and 3c) and propyne by Sonogashira coupling (Figure 4 and 5) [12], [13]. A di-butylaminomethylidene group was used to protect the exocyclic amine of 3c instead of an acetyl or benzoyl group in order to avoid a potential cyclization side-reaction involving the amide and the 5-propynyl group [14]. The presence of a propynyl group in 4u and 5c was confirmed by the characteristic chemical shift (4–5 ppm) of the methyl carbon in 13C NMR [15] together with 1H-NMR and ESI-MS analysis. gUpropTP and gCpropTP (1u and 1c) were synthesized from the corresponding nucleosides (5u and 6c) using the one-pot, salicylchlorophosphorin approach developed by Ludwig and Eckstein [16]. The final purified products 1u and 1c were characterized by 1H- and 31P-NMR and by ESI-MS. In addition, 1u and 1c have similar UV absorption profiles to those reported for 5-propynyl-deoxyribonucleosides [15].

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