Thermodynamic contributions for the inclusion of bulges and mismatches in dna hairpin loops

Bulges and mismatches in DNA can promote frame-shift and translational/transversional mutations, respectively. A complete characterization of the effects that bulges and mismatches have on duplex stability is required to understand the mechanisms of mutations. In this work, we used a combination of temperature-dependent UV spectroscopy, differential scanning calorimetry and circular dichroism techniques to investigate the thermodynamic contribution and conformation for the incorporation of bulges and mismatches in duplex DNA. Specifically, we obtained standard thermodynamic profiles (δG, δH, and δS) for the helix-coil transition of a host DNA hairpin-loop with sequence d(GTAGCT₅GCTAC), T5 is a loop of 5 thymines; and hairpins containing one of the four potential bulges (dC, dT, dA, or dG) and/or one of the homo-mismatches (dC-dC, dT-dT, dA-dA, or dG-dG) in two base-pair stack environments (TA/TA and AG/CT) located in the stem of the host hairpin. All hairpins melted in two-state monophasic transitions with transition temperatures, T_M, independent of strand concentration, which is consistent with their intramolecular formation. Their CD spectra indicated all hairpins folded in the "B" conformation. However, the incorporation of a lesion resulted in lower thermal (δT_M = 6.6 to 24.7 C) and thermodynamic (δδG = 0.1 to 3.0 kcal/mol) stabilities, due to lower enthalpies (δδH = 0.2 to 14.7 kcal/mol), indicating a loss of base-pair stacking interactions. Overall, bulges cause higher destabilization than mismatches. Lesions incorporated in the AG/CT environment generated slightly larger destabilization effect relative to the TA/TA environment. The results show that cytosine lesions destabilized the most followed by adenine lesions, thymine lesions, and guanine lesions.