光機能性修飾核酸の合成とDNA中メチル化シトシンに選択的な光一電子酸化反応

抄録

Cytosine methylation at the 5-position in DNA has been implicated in a epigenetic regulation of genetic information and diseases including cancer, and therefore it has become important to identify the methylation status of a specified cytosine residue in DNA to obtain a molecular insight into the biological function of cytosine methylation. To discriminate between cytosine and 5-methylcytosine (^mC), we investigated a photochemical one-electron oxidation at 5-methylcytosine in DNA, using oligodeoxynucleotides (ODN) possessing a 2-methyl-1,4-naphthoquinone (NQ) chromophore. Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of the NQ-tethered ODN and its complementary strand bearing ^mC led to oxidative cleavage at the ^mC residue, which is located at the front of the NQ chromophore. Detailed mechanistic study revealed that rapid deprotonation of the ^mC radical cation generated by NQ-photosensitization and subsequent addition of molecular oxygen to generate alkali labile oxidized form would lead to cleavage at the original ^mC site. We also designed an efficient fluorometric detection system of DNA methylation based on a combination of photooxidative DNA cleavage reaction with NQ chromophore and invasive cleavage reaction with Human Flap endonuclease-I. Enzymatic treatment of a mixture of photochemically fragmented target ODNs at ^mC and hairpin-like probe oligomer possessing a fluorophore and a quencher resulted in a dramatic enhancement of fluorescence. Thus, the presented systems would be a highly sensitive protocol for the identification of methylation status in DNA.