Nucleobase recognition by hydrogen bond forming ligands and its use for fluorescence detection of single-nucleotide polymorphisms

In combination with abasic site (AP site)-containing oligodeoxynucleotides (ODNs), we demonstrate the potential use of hydrogen bond-forming ligands, 2-amino-7-methyl-1,8-naphthyridine (AMND) and 2-amino-4-oxopteridine (pterin), for the recognition of nucleobases in the aqueous phase. Our strategy is based on the construction of an AP site in ODN duplexes, which allows small synthetic and/or biotic ligands to strongly bind to target nucleobases; an A-P 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, From an examination of the binding behaviors by melting temperature (T.), fluorescence and circular dichroism spectra measurements, it has been found that AMND selectively binds to a cytosine base with a 1 : 1 binding constant of >10(6) M-1, while pterin shows selectivity for a guanine base (K-11 = 1.2 x 10(4) M-1) over other nucleobases. The observed high stabilities of 1 : 1 complexes can be explained by a cooperative binding event, i.e., hydrogen-bonding with target nucleobases and stacking with nucleobases flanking the AP site. The potential use of these ligands is also presented for the fluorescence detection of single-nucleotide polymorphisms (SNPs) of the cancer repression gene p53.