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, Pivoňková, Hana, Kalachova, Lubica, Černocká, Hana, Cahová, Hana, Pohl, Radek, Šebest, Peter, Havran, Luděk, Hocek, Michal, Fojta, Miroslav
• Format: Article
• Language: English
• Subjects: DNA polymerase; electrochemistry; oligonucleotides; osmium; ruthenium
• ISSN: 0947-6539; 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 Ru2+ or Os2+ complexes. Square‐wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru2+/3+ or Os2+/3+. The redox potentials of the Ru2+ complexes (1.1–1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os2+ complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru2+‐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. Metal‐labeled DNA: DNA‐bearing [Ru(bpy)3]2+ and [Os(bpy)3]2+ complexes have been constructed by polymerase incorporation of the corresponding base‐modified nucleoside triphosphates (see figure). The Ru‐modified DNA was luminescent, while the Os complexes could be detected by electrochemistry. A complete four‐color redox labeling of DNA has been developed and used for minisequencing.