Fluorescence detection of cytosine/guanine transversion based on a hydrogen bond forming ligand

In combination with abasic site (AP site)-containing oligodeoxynucleotides (ODNs), we demonstrate potential use of a hydrogen bond forming ligand, 2-amino-7-methyl-1,8-naphthyridine (AMND), for the fluorescence detection of the cytosine (C)/guanine (G) mutation sequence of the cancer repression gene p53. Our method is based on construction of the AP site in ODN duplexes, which allows small synthetic ligands to bind to target nucleobases accompanied by fluorescence signaling: an AP site-containing ODN is hybridized with a target ODN so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleobases through hydrogen-bonding. In 10 mM sodium cacodylate buffer solutions (pH, 7.0) containing 100 mM NaCl and 1.0 mM EDTA, AMND is found to strongly bind to C (Kd=1.5×10−6 M) in the target ODN while the binding affinity for G is relatively moderate (Kd=50×10−6 M). Significant fluorescence quenching of AMND is observed only when binding to C, making it possible to judge the C/G transversion with the naked eye.