A Pyrazine-based Fluorescence-enhancing Ligand with a High Selectivity for Thymine in AP Site-containing DNA Duplexes

A fluorescent pyrazine derivative, 3,5-diamino-6-chloro-2-pyrazine carbonitrile (DCPC), is presented as a promising light-up ligand for single-nucleotide polymorphisms (SNPs) typing. In solutions buffered to pH 7.0 (7 = 0.11 M, at 5°C), DCPC can bind to thymine selectively over other nucleobases opposite an abasic site in DNA duplexes (5’-GTGTG CGTTG ANA TGGAC GCAGA-373’-CACAC GCAAC TXT ACCTG CGTCT-5’, X = abasic site, N = target nucleotide) with a dissociation constant of 2.6 μM. The binding of DCPC is accompanied by a significant enhancement of its fluorescence (λmax, 412 nm), and the response is highly selective to thymine base. These binding and sensing properties allow a clear detection of thymine-related mutations present in polymerase chain reaction (PCR) amplification products.