Nucleic Acids Research Advance Access originally published online on June 26, 2008
Nucleic Acids Research 2008 36(13):4257-4265; doi:10.1093/nar/gkn404
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Nucleic Acids Research, 2008, Vol. 36, No. 13 4257-4265
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chemistry and Synthetic Biology |
Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleosides
1Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, Gunma 376-8515, 2PRESTO, Japan Science and Technology Agency (JST), Chiyodaku, Tokyo 102-0075 and 3Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
*To whom correspondence should be addressed. Tel/Fax: +81 277 30 1222; Email: kuwahara{at}chem-bio.gunma-u.ac.jp
Received April 27, 2008. Revised June 7, 2008. Accepted June 9, 2008.
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.