One-electron photooxidation and site-selective strand cleavage at 5-methylcytosine in DNA by sensitization with 2-methyl-1,4-naphthoquinone-tethered oligonucleotides

Photosensitized one-electron oxidation was applied to discriminate a specific base site of 5-methylcytosine (C-m) generated in DNA possessing a partial sequence of naturally occurring p53 gene, using a sensitizing 2-methyl-1,4-naphthoquinone (NQ) chromophore tethered to an interior of oligodeoxynucleotide (ODN) strands. Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of C-m-containing DNA and NQ-tethered complementary ODN led to oxidative strand cleavage selectively at the C-m site, when the NQ chromophore was arranged so as to be in close contact with the target C-m. The target C-m is most likely to be one-electron oxidized into the radical cation intermediate by the sensitization of NQ. The resulting C-m radical cation may undergo rapid deprotonation and subsequent addition of molecular oxygen, thereby leading to its degradation followed by strand cleavage at the target C-m site. In contrast to C-m-containing ODN, ODN analogs with replacement of normal cytosine, thymine, adenine, or guanine at the C-m site underwent less amount of such an oxidative strand cleavage at the target base site, presumably due to occurrence of charge transfer and charge recombination processes between the base radical cation and the NQ radical anion. Furthermore, well designed incorporation of the NQ chromophore into an interior of ODN could suppress a competitive strand cleavage at consecutive guanines, which occurred as a result of positive charge transfer. Thus, photosensitization by an NQ-tethered ODN led to one-electron oxidative strand cleavage exclusively at the target C-m site, providing a convenient method of discriminating C-m in naturally occurring DNA such as human p53 gene as a positive band on a sequencing gel.