Use of vitamin B2 for fluorescence detection of thymidine-related single-nucleotide polymorphisms

In combination with abasic site (AP site)-containing DNAs, potential use of a biotic fluorescence compound, Vitamin B, (riboflavin), is demonstrated for the fluorescence detection of the thymine (T)-related single-nucleotide polymorphisms. Our method is based on construction of the AP site in DNA duplexes, which allows small ligands to bind to target nuclectides accompanied by fluorescence signaling: an AP site-containing probe DNA is hybridized with a target DNA so as to place the AP site toward a target nucleobase, by which hydrophobic microenvironments are provided for ligands to recognize target nucleotides through stacking and hydrogen-bonding interactions. In 10 mM sodium cacodylate buffer solutions (pH 7.0) containing 100mM NaCl and 1.0mM EDTA, Vitamin B, is found to selectively bind to T (K-11 = 1.8 x 10(6) M-1 at 5 degrees C) over other nucleobases, and this is accompanied by significant quenching of its fluorescence. While the sensing functions depend on the flanking sequences to the AP site, Vitamin B-2 is applicable to the detection of T/C (cytosine), T/G (Panine) and T/A (adenine) mutation sequences of the CYP2A6 gene, where the flanking nucleobases are guanines in both positions (-G (X) under barG-, (X) under bar = AP site). (c) 2005 Elsevier B.V. All rights reserved.