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

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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.

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

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