Discrimination between 8-oxo-2′-deoxyguanosine and 2′-deoxyguanosine in DNA by the single nucleotide primer extension reaction with adap triphosphate

The adenosine derivative of 2-oxo-1,3-diazaphenoxazine (Adap) exhibits a superb ability to recognize and form base pairs with 8-oxo-2′-deoxyguanosine (8-oxo-dG) in duplex DNA. In this study, the triphosphate of Adap (dAdapTP) was synthesized and tested for single nucleotide incorporation into primer strands using the Klenow Fragment. The efficiency of dAdapTP incorporation into 8-oxo-dG-containing templates was more than 36-fold higher than with dG-containing templates, and provides better discrimination than does the incorporation of natural 2′-deoxyadenosine triphosphate (dATP). The selective incorporation of dAdapTP into 8-oxo-dG templates was therefore applied to the detection of 8-oxo-dG in human telomeric DNA sequences extracted from H₂O₂-treated HeLa cells. The enzymatic incorporation of dAdapTP into 8-oxo-dG-containing templates may provide a novel basis for sequencing oxidative DNA damage in the genome. One is not like the other: The triphosphate of Adap (dAdapTP) was synthesized and used in single nucleotide primer extension reactions with the Klenow Fragment. Based on the extension efficiency, dAdapTP showed excellent discrimination between 8-oxo-dG and dG in the template ODNs. The sequence-specific detection of 8-oxo-dG in human telomeric DNA sequences extracted from H₂O₂-treated HeLa cells was also performed by an enzymatic incorporation reaction with dAdapTP.