Published online 27 November 2005
Article |
Synthesis of novel poly(dG)–poly(dG)–poly(dC) triplex structure by Klenow exo– fragment of DNA polymerase I
1Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University Ramat Aviv, 69978 Israel 2Nanotechnology Center, Tel Aviv University Ramat Aviv, 69978 Israel 3Laboratory for the Physics of Nanostructures, Ecole Polytechnique Fédérale de Lausanne CH-1015 Lausanne, Switzerland 4Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences 117871 Moscow, Russia
*To whom correspondence should be addressed. Tel: +972 3 640 7138; Fax: +972 3 640 6834; Email: s2shak{at}post.tau.ac.il
Received August 31, 2005. Revised October 31, 2005. Accepted October 31, 2005.
The extension of the G-strand of long (700 bp) poly(dG)–poly(dC) by the Klenow exo– fragment of DNA polymerase I yields a complete triplex structure of the H-DNA type. High-performance liquid chromatography analysis demonstrates that the length of the G-strand is doubled during the polymerase synthesis. Fluorescence resonance energy transfer analysis shows that the 5' ends of the G- and the C-strands, labeled with fluorescein and TAMRA, respectively, are positioned close to each other in the product of the synthesis. Atomic force microscopy morphology imaging shows that the synthesized structures lack single-stranded fragments and have approximately the same length as the parent 700 bp poly(dG)–poly(dC). CD spectrum of the polymer has a large negative peak at 278 nm, which is characteristic of the poly(dG)–poly(dG)–poly(dC) triplex. The polymer is resistant to DNase and interacts much more weakly with ethidium bromide as compared with the double-stranded DNA.