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.
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.
Vrábel
Milan
M
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead Sciences and IOCB Research Center, Flemingovo nam. 2, 16610 Prague 6, Czech Republic.
Horáková
Petra
P
Pivonková
Hana
H
Kalachova
Lubica
L
Cernocká
Hana
H
Cahová
Hana
H
Pohl
Radek
R
Sebest
Peter
P
Havran
Ludek
L
Hocek
Michal
M
Fojta
Miroslav
M
D003116:Color; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D004563:Electrochemistry; D049449:Luminescence; D009841:Oligonucleotides / Q000737:chemistry; D009992:Osmium / Q000737:chemistry*; D010084:Oxidation-Reduction; D012428:Ruthenium / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D009992:Osmium; D012428:Ruthenium; D004247:DNA; D004259:DNA-Directed DNA Polymerase