Our laboratory is interested in predicting the thermal stability and melting behavior of nucleic acids from knowledge of their sequence. One focus is to understand how sequence, duplex and triplex stabilities, and solution conditions affect the melting behavior of complex DNA structures, such as intramolecular DNA complexes containing triplex and duplex motifs. For these reasons, in this chapter, we used a combination of UV and circular dichroism (CD) spectroscopies and differential scanning calorimetry (DSC) techniques to obtain a full thermodynamic description of the melting behavior of six intramolecular DNA complexes with joined triplex and duplex motifs. The CD spectra at low temperatures indicated that these complexes maintained the "B" conformation. UV and DSC melting curves of each complex show biphasic or triphasic transitions. However, their corresponding transition temperatures (T(m)s) remained constant with increasing strand concentration, confirming their intramolecular formation. Deconvolution of the DSC thermograms allowed us to determine standard thermodynamic profiles for the transitions of each complex. For each transition, the favorable free energy terms result from the characteristic compensation of a favorable enthalpy and unfavorable entropy contributions. The magnitude of these thermodynamic parameters (and associated T(m)s) indicate that the overall folding of each complex depends on several factors: (a) the extent of the favorable heat contributions (formation of base-pair and base-triplet stacks) that are compensated with both the ordering of the oligonucleotide and the putative uptake of protons and ions; (b) inclusion of the more stable C(+)GC base triplets; (c) stabilizing the duplex stem of the complex; and (d) solution conditions, such as pH and salt concentration. Overall, the temperature-induced unfolding of each complex corresponds to the initial disruption of the triplex motif (removal of the third strand) followed by the partial or full unfolding of the duplex stem.
|Original language||English (US)|
|Number of pages||26|
|Journal||Methods in enzymology|
|Publication status||Published - Dec 1 2009|
ASJC Scopus subject areas
- Molecular Biology