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Examination of the Premelting Transition of DNA A-Tracts Using a Fluorescent Adenosine Analogue

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journal contribution
posted on 18.04.2006, 00:00 by Katherine E. Augustyn, Kristi Wojtuszewski, Mary E. Hawkins, Jay R. Knutson, Ishita Mukerji
The fluorescent adenosine analogue 4-amino-8-(2-deoxy-β-d-ribofuranosyl)-5‘-O-dimethoxytrityl-6-methyl-7(8H)-pteridone (6MAP) has been used to perform residue specific analyses of DNA A-tracts during the premelting transition. DNA A-tracts, which exhibit sequence-induced curvature, adopt a B-DNA conformation as a function of increasing temperature. Fluorescence melting curves indicate that 6MAP is a more sensitive reporter of the premelting transition than UV absorption spectroscopy. Further, residue specific fluorescence analyses of A-tract and control duplexes reveal that some of the conformational changes associated with the premelting transition occur within A-tract regions. Analyses of the energetics of the premelting transition indicate that ApA steps make a larger enthalpic contribution to the premelting transition than ApT steps. To explore the effect of cations on the premelting transition, fluorescence melts were performed in the presence of NH4+, Mg2+, and low (0.05 M) and high (0.5 M) concentrations of Na+. These studies show that the fluorescence intensity changes associated with the premelting transition are sensitive to cation type and concentration and are larger and more pronounced in the presence of 0.5 M Na+, NH4+, and Mg2+. Incorporation of 6MAP into longer duplexes containing phased A-tracts shows that the local environment of adenosines in phased A-tracts is similar to that of individual A-tracts. Fluorescence quenching results indicate that ApA and ApT steps within A-tracts are less solvent exposed than their counterparts in control sequence isomers, possibly because of the narrowed minor groove of A-tract sequences.