Guanine and 8-Azaguanine in Anomeric DNA Hybrid Base Pairs: Stability, Fluorescence Sensing, and Efficient Mismatch Discrimination with alpha-D-Nucleosides

Abstract

The alpha-anomers of 8-aza-2'-deoxyguanosine (alpha G(d)*) and 2'-deoxyguanosine (alpha G(d)) were site-specifically incorporated in 12-mer duplexes opposite to the four canonical DNA constituents alpha G(d)*, dG, dT, and dC. Oligodeoxyribonucleotides containing aGd* display significant fluorescence at slightly elevated pH (8.0). Oligodeoxyribonucleotides incorporating beta-anomeric 8-aza-2'-deoxyguanosine (G(d)*) and canonical dG were studied for comparison. For alpha G(d)* synthesis, an efficient purification of anomeric 8-azaguanine nucleosides was developed on the basis of protected intermediates, and a new alpha G(d)* phosphoramidite was prepared. Differences were observed for sugar conformations (N vs S) and pK(a) values of anomeric nucleosides. Duplex stability and mismatch discrimination were studied employing UV-dependent melting and fluorescence quenching. A gradual fluorescence change takes place in duplex DNA when the alpha-nucleoside alpha G(d)* was positioned opposite to the four canonical beta-nucleosides. The strongest fluorescence decrease appeared in duplexes incorporating alpha G(d)*-Cd base pair matches. Decreasing fluorescence corresponds to increasing T-m values. For mismatch discrimination, the alpha-anomers alpha G(d)* and aGd are more efficient than the corresponding beta-nucleosides. Duplexes with single ``purine-purine'' alpha G(d)*-alpha G(d)* or alpha G(d)-alpha G(d) base pairs are significantly more stable than those displaying beta-D configuration. CD spectra indicate that single mutations by alpha-anomeric nucleosides do not affect the global structure of B-DNA.