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

The α-anomers of 8-aza-2'-deoxyguanosine (αGd *) and 2'-deoxyguanosine (αGd ) were site-specifically incorporated in 12-mer duplexes opposite to the four canonical DNA constituents dA, dG, dT, and dC. Oligodeoxyribonucleotides containing αGd * display significant fluorescence at slightly elevated pH (8.0). Oligodeoxyribonucleotides incorporating β-anomeric 8-aza-2'-deoxyguanosine (Gd *) and canonical dG were studied for comparison. For αGd * synthesis, an efficient purification of anomeric 8-azaguanine nucleosides was developed on the basis of protected intermediates, and a new αGd * phosphoramidite was prepared. Differences were observed for sugar conformations ( N vs S) and p Ka 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 α-nucleoside αGd * was positioned opposite to the four canonical β-nucleosides. The strongest fluorescence decrease appeared in duplexes incorporating αGd *-Cd base pair matches. Decreasing fluorescence corresponds to increasing Tm values. For mismatch discrimination, the α-anomers αGd * and αGd are more efficient than the corresponding β-nucleosides. Duplexes with single "purine-purine" αGd *-αGd * or αGd -αGd base pairs are significantly more stable than those displaying β-d configuration. CD spectra indicate that single mutations by α-anomeric nucleosides do not affect the global structure of B-DNA.