Lawrence Berkeley National Laboratory

Abasic site (AP)-containing duplexes, in which the AP site was flanked by adenine (A) or cytosine (C) bases had been shown to be more stable with flanking A than with C bases [J. S gi, B. Hang and B. Singer (1999) Chem. Res. Toxicol. 12, 917-923]. In this work we investigated whether the smaller destabilization by an AP site, when the neighbors are A, is a general effect of the purine neighbors versus the pyrimidines. An AP site (x), flanked by symmetric doublet bases, was first incorporated opposite T into 15-mer duplexes. Duplex stability was markedly decreased by the AP site in all cases, as compared to the control duplexes. The largest destabilization was observed with the duplex containing a central -TTxTT- sequence in which the DTm was -19.4oC and the DDGo37 was -6.57 kcal/mol. Changing the central -TTxTT- to the -AAxAA- sequence resulted in a smaller destabilization (-17.3 oC and -5.65 kcal/mol), although the neighbors were AoT base pairs in both cases. Similarly, the AP site in -GGxGG- sequence proved to be less destabilizing (-13.9oC, -5.74 kcal/mol) than in the -CCxCC- sequence (-15.8oC, -7.39 kcal/mol) although G.C pairs flanked the AP site in both cases. The same differential destabilization was also observed with duplexes that contained an A or C opposite the AP site. The average stabilizing effect of the symmetric doublet purine neighbors of an AP site opposite a T, A or C was 3.2oC DTm and 1.3 kcal/mol DDGo37, as compared to the pyrimidine neighbors. The opposite G base reduced or eliminated the differential effect of the neighbors. Unrestrained molecular dynamics was used to evaluate the effect of the flanking sequences on structural features of the duplexes containing T opposite the AP site. Explicit solvent and the particle mesh Ewald method were applied for accurate representation of the electrostatic interactions. The duplexes with doublet pyrimidine neighbors showed a larger magnitude of curvature around the lesion site than did the duplexes wit