Ultraviolet absorption techniques were used to study the thermodynamics of duplex formation for a DNA decamer, d(GCGAAAAGCG).d(CGCTTTTCGC), and a series of related duplexes, each of which contains a bulged base centered in the A.T tract. Thermodynamic parameters were obtained from nonlinear least-squares fits of the melting curves and the concentration dependences of the melting temperatures. Duplexes containing a localized single-base bulge were found to be 3.5-4.6 kcal/mol less stable than the decamer at 37 degrees C. These results indicate that both the identity of the bulged base and the strand in which it is located may influence the amount by which the duplex is destabilized. Bulged bases located in the T-strand, d(CGCTTYTTCGC), in position Y, were observed to be slightly more destabilizing than those located in the A-strand, d(GCGAAXAAGCG), in position X. Bulged purines may be more destabilizing than bulged pyrimidines.

One- and two-dimensional NMR, UV absorption experiments, and molecular mechanics calculations were conducted on an oligonucleotide duplex (dGCGAATAAGCG)2 which will be referred to as the T-11-mer. This oligonucleotide forms a duplex that is primarily B-form and contains two adjacent G.A and A.A base pairs and two 3' unpaired guanosines. The adjacent mismatch base pairs have an unusual structure which includes overwinding the helix and stacking with the base from the complementary strand (A4 with A8 and G3 with A7) instead of stacking with the base which is sequential on the strand. The exchangeable and nonexchangeable proton NMR spectra of the duplex have been characterized in H2O and D2O solution at neutral and acidic pH. The duplex is stabilized upon protonation; however, no additional hydrogen bonds are formed. We have observed the amino protons of adenosines A4 and A8 and guanosine G3 as a function of temperature and pH. These amino protons are involved in hydrogen bonds with the purine N3 or N7 acting as acceptors. Through the observation of a variety of NOE signals, the structure of the G.A and A.A mismatch base pairs has been defined.

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