Book of Abstracts: Albany 2005
Melting Behavior of DNA Triplexes of the Pyrimidine Motif
Our laboratory is interested in predicting the thermal stability and melting behavior of nucleic acids from knowledge of its sequence; specially, the unfolding of intramolecular triplexes of the ?Pyrimidine? motif. Our main objective is to understand how sequence, strand polarity, duplex stability, and solution conditions affects the melting behavior of these triplexes. In this work, we use a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry (DSC) techniques to investigate the unfolding of intramolecular triplexes with the following sequences: AnC5TnC5Tn (n = 5-9), A(GA)nC5T(CT)nC5T(CT)n (n = 2-3), T6C5T6C5A6, A6GCGCT5GCGCT6C5T6, A6GCGCT5GCGCT6C5T6C, AGAGA2GCGCT5GCGCT2CTCTC5TC-TCT2 and T2CTCTC5TCTCT2CGCGT5CGCGA2GAGA, bases in italics correspond to end loops. UV and DSC melting profiles of these molecules are complex, some triplexes melted in a monophasic, transition while others in biphasic or triphasic transitions. However, the corresponding transition temperatures, TM?s, remained constant with increasing strand concentration, confirming their intramolecular formation. Deconvolution of the DSC curves allowed us to determine standard thermodynamic profiles for each transition. The favorable free energy terms result from the characteristic enthalpy-entropy compensation. The magnitude of the thermodynamic parameters indicate that the overall unfolding of each triplex depends on several factors: a) the extent of the favorable heat contributions (formation of base triplet stacks) that are compensated with both the ordering of the oligonucleotide and the putative uptake of protons ions and water molecules; b) inclusion of the more stable C+GC base triplets; c) stabilizing the duplex portion of the triplex; and d) solution conditions, such as pH and salt concentration.
Departments of Pharmaceutical Sciences