Book of Abstracts: Albany 2003
June 17-21 2003
Physical Chemistry of the Unfolding of DNA Intramolecular Triplexes
DNA oligonucleotides may be used in antigene therapies by targeting duplex DNA, via triplex formation, to control gene expression. In this work, we have investigated the energetic and hydration contributions for the substitution of cytosine with 5-methyl-cytosine in nucleic acid triplexes. We have used a combination of optical and calorimetric techniques to determine complete thermodynamic profiles for the unfolding of two oligomers with the following sequences: d(AGAGAC5TCTCTC5TCTCT), d(AGAGAC5TCTCTC5TMe-CTMe-CT), and control sequence d(AGAGAC5TCTCTC2). The unfolding of each DNA oligomer takes place in monophasic transitions and their transition temperatures, TM, are independent of the strand concentration, which confirms the formation of intramolecular complexes: two triplexes with double loops and a duplex with a single loop. The substitution of two cytosines for two 5-methyl-cytosines yielded more stable triplexes, by ~ 5° C (ΔΔG°cal = 1 kcal/mol). The unfolding of each triplex results from the characteristic compensation of an unfavorable enthalpy with a favorable entropy contribution. The overall unfavorable enthalpy contribution yielded an average of 23.2 kcal/mol for an TAT/C+GC (or C+GC/TAT) base-triplet stack. At pH 5.2, the TM of each triplex increases as the salt concentration is increased, however, this trend is reversed at pH 7.0. This effect yields a small release of counterions (~0.37 mol Na+/mol triplex). The TM of each triplex increases as the pH is decreased yielding a net release of ~2.7 mol H+/mol triplex and 0.36 mol H+/mol duplex. This is consistent with the required protonation of two cytosines at the stem and of the loop cytosines. Furthermore, the TM also increases as the activity of water is increased yielding a large release of ~33 water molecules per triplex, which indicates that the folded triplexes are more hydrated. The favorable entropy contribution in the unfolding of these intramolecular triplexes is due to the release of ions, protons and water molecules. The overall conclusion is that the 5-methyl-cytosine/cytosine substitution stabilizes a nucleic acid triplex, yielding similar thermodynamic profiles in their unfolding reactions.
Department of Pharmaceutical Sciences