Base-modified DNA labeled by [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes: construction by polymerase incorporation of modified nucleoside triphosphates, electrochemical and luminescent properties, and applications

• Published in: Chemistry : a European journal 2009-01, Vol.15 (5), p.1144-1154
• Main Authors: Vrábel, Milan, Horáková, Petra, Pivonková, Hana, Kalachova, Lubica, Cernocká, Hana, Cahová, Hana, Pohl, Radek, Sebest, Peter, Havran, Ludek, Hocek, Michal, Fojta, Miroslav
• Format: Article
• Language: English
• Subjects: Color; Cross-Linking Reagents - chemistry; DNA - chemistry; DNA-Directed DNA Polymerase - chemistry; Electrochemistry; Luminescence; Oligonucleotides - chemistry; Osmium - chemistry; Oxidation-Reduction; Ruthenium - chemistry; Staining and Labeling - methods
• ISSN: 1521-3765
• DOI: 10.1002/chem.200801538

Modified 2'-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru(2+) or Os(2+) complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru(2+/3+) or Os(2+/3+). The redox potentials of the Ru(2+) complexes (1.1-1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os(2+) complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru(2+)-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for "multicolor" redox labeling of DNA and for DNA minisequencing.