Recognition of a C–C Mismatch in a DNA Duplex Using a Fluorescent Small Molecule with Application for “Off–On” Discrimination of C/G Mutation

Abstract

The fluorescent small molecule 2-amino-7-methyl-1,8-naphthyridine (AMND) can selectively bind to a cytosine (C) at a C–C mismatch in double-stranded DNA (dsDNA). The interactions between AMND and C–C mismatch-containing dsDNA were investigated by measuring ultraviolet (UV) absorption as a function of temperature to obtain melting curves as well as circular dichroism and fluorescence spectra. Results show that AMND strongly stabilizes C–C mismatch-containing dsDNA, whereas fully matched duplexes are not stabilized under the same conditions. The fluorescence of AMND was efficiently quenched when it was bound to a C–C mismatch in dsDNA. Binding constants (K₁₁), obtained by fluorescence titration, were 1.2 × 10⁵ M⁻¹. Although sensing functions depend on the sequences flanking the mismatch site, the change in AMND fluorescence intensity can be utilized to detect the C–C mismatch-containing dsDNA. Accordingly, discrimination of the C/G mutation in the model sequence (PGR gene rs1255998) was achieved by visualizing fluorescence of AMND. A probe DNA molecule was designed to contain a C opposite the C/G base in the target DNA, and this probe was used to hybridize the target DNA. The fluorescence of AMND was “on” for a C–G match, while the fluorescence was “off” for a C–C mismatch. This assay is simple and does not require DNA labeling.