Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleosides.
In order to systematically analyze the effects of nucleoside modification of sugar moieties in DNA polymerase reactions, we synthesized 16 modified templates containing 2',4'-bridged nucleotides and three types of 2',4'-bridged nucleoside-5'-triphospates with different bridging structures. Among the five types of thermostable DNA polymerases used, Taq, Phusion HF, Vent(exo-), KOD Dash and KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modified templates containing 2'-O,4'-C-methylene-linked nucleotides at intervals of a few nucleotides, even at standard enzyme concentrations for 5 min. Although the Vent(exo-) DNA polymerase also read through these modified templates, kinetic study indicates that the KOD(exo-) DNA polymerase was found to be far superior to the Vent(exo-) DNA polymerase in accurate incorporation of nucleotides. When either of the DNA polymerase was used, the presence of 2',4'-bridged nucleotides on a template strand substantially decreased the reaction rates of nucleotide incorporations. The modified templates containing sequences of seven successive 2',4'-bridged nucleotides could not be completely transcribed by any of the DNA polymerases used; yields of longer elongated products decreased in the order of steric bulkiness of the modified sugars. Successive incorporation of 2',4'-bridged nucleotides into extending strands using 2',4'-bridged nucleoside-5'-triphospates was much more difficult. These data indicate that the sugar modification would have a greater effect on the polymerase reaction when it is adjacent to the elongation terminus than when it is on the template as well, as in base modification.
Kuwahara
Masayasu
M
Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Gunma 376-8515, Japan. kuwahara@chem-bio.gunma-u.ac.jp
Obika
Satoshi
S
Nagashima
Jun-ichi
J
Ohta
Yuki
Y
Suto
Yoshiyuki
Y
Ozaki
Hiroaki
H
Sawai
Hiroaki
H
Imanishi
Takeshi
T
D001952:Bridged-Ring Compounds / Q000737:chemistry; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007700:Kinetics; D009705:Nucleosides / Q000737:chemistry; D009711:Nucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D011122:Polyphosphates / Q000737:chemistry; D013698:Templates, Genetic
D001952:Bridged-Ring Compounds; D017931:DNA Primers; D009705:Nucleosides; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D011122:Polyphosphates; D004247:DNA; D004259:DNA-Directed DNA Polymerase