Unfolding thermodynamics of DNA intramolecular complexes involving joined triple- and double-helical motifs.

Irine Khutsishvili, Sarah Johnson, Hui Ting Lee, Luis A. Marky

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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 languageEnglish (US)
Pages (from-to)477-502
Number of pages26
JournalMethods in enzymology
Volume466
StatePublished - Dec 1 2009

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Thermodynamics
Freezing
Differential Scanning Calorimetry
Melting
Differential scanning calorimetry
DNA
Circular Dichroism
Temperature
Hot Temperature
Circular dichroism spectroscopy
Transition Temperature
Dichroism
Entropy
Deconvolution
Oligonucleotides
Base Pairing
Nucleic Acids
Free energy
Conformations
Protons

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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Unfolding thermodynamics of DNA intramolecular complexes involving joined triple- and double-helical motifs. / Khutsishvili, Irine; Johnson, Sarah; Lee, Hui Ting; Marky, Luis A.

In: Methods in enzymology, Vol. 466, 01.12.2009, p. 477-502.

Research output: Contribution to journalArticle

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