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


RESULTS
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.
Chen
Jesse J
JJ
Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.
Tsai
Ching-Hsuan
CH
Cai
Xin
X
Horhota
Allen T
AT
McLaughlin
Larry W
LW
Szostak
Jack W
JW
D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005990:Glycerol / Q000737:chemistry*; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D013698:Templates, Genetic
D017931:DNA Primers; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005990:Glycerol