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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

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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 (K 11), 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.

Keywords: 2-Amino-7-methyl-1,8-naphthyridine; Circular dichroism; C‐C mismatch; Fluorescence spectroscopy; Recognition; Single base mutation

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/11-06410

Affiliations: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Materials Science, Shaanxi Normal University, Xi'an 710062, China

Publication date: February 1, 2012

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