A THERMODYNAMIC INVESTIGATION OF THE MELTING OF B-Z JUNCTION FORMING DNA OLIGOMERS

Ultraviolet absorbance methods were used to characterize the thermodyn amics of melting of a series of 16 bp deoxyoligonucleotides over a wid e range of NaCl concentrations (0-4.5 M) and to obtain complete thermo dynamic profiles for their melting at 0.115 and 4.5 M NaCl The sequenc e of the series (one strand of duplex) was: 5'-CGCGCGCGAMNGACTG-3', wh ere C indicates m(5)dC and -MN- was varied to include all combinations of Py:Py stacks (CC, TT, CT, TC). The unmethylated deoxyoligonucleoti de 5'-CGCGCGCGACTGACTG-3' was used as a control sequence. All of the m ethylated oligonucleotides studied undergo a NaCl-induced transition t o a hybrid form containing a left-handed, Z-DNA, region joined to a ri ght-handed region by a B-Z junction. Our experiments allowed us to qua ntitatively evaluate the effects of NaCl, sequence, and methylation an d the transition to the hybrid BZ structure on DNA thermal stability. We found that alteration of a single dinucleotide step has profound ef fects on the thermal stabilities of the 16 bp fragments studied. Methy lation was found to destabilize the double helix, resulting in a decre ase in T-m. Transition to the hybrid BZ structure, somewhat Surprising ly, was found to only slightly destablize DNA, with an observed decrea se in free energy of melting of approximately 0.5 kcal/mol relative to the control, right-handed, sequence in high salt. Transition melting temperatures es (T-m) were found, in agreement with previous studies o n polymeric DNA, to depend upon NaCl concentration in a complicated, n onlinear fashion. T-m values increase td maximal values at circa 1.0 M NaCl, but decrease thereafter with further addition of salt. Our expe riments provide quantitative data that help to describe the factors co ntributing to the stability of the DNA double helix and that document the ability of DNA to accommodate an unusual structure, the B-Z juncti on, with little energetic cost to its stability.