Analytical Sciences

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

Chunxia ZHAO, Arivazhagan RAJENDRAN, Qing DAI, Seiichi NISHIZAWA, and Norio TERAMAE

Department of Chemistry, Graduate School of Science, Tohoku University, and CREST, Japan Science and Technology Agency (JST), Aoba, Sendai 980-8578, Japan

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 (I = 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-3′/3′-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 (λ_max, 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.

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