Albany 2013: Book of Abstracts

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Conversation 18
June 11-15 2013
©Adenine Press (2012)

PPC Measurements of Unfolding Volumes of DNA Stem-Loop Motifs

One focus of our research is to further our understanding of the physico-chemical properties of non-canonical nucleic acid structures. In this work, DNA hairpins are used to mimic a common motif present in RNA, i.e., a stem-loop motif with a bulge or internal loop in their stem. Specifically, we used a combination of temperature-dependent UV spectroscopy, differential scanning (DSC) and pressure perturbation (PPC) calorimetric techniques to determine complete thermodynamic profiles for the helix-coil transitions of two sets of hairpins with 5’-3’ sequences: d(GCGCTnGTAACT5GTTACGCGC) and d(GCGCTnGTAACT5GTTACTnGCGC). “Tn” is a variable loop of thymines, n = 1, 3 or 5; and “T5” is an end loop of five thymines. Unfolding curves show monophasic transitions with TMs independent of strand concentration, confirming their intramolecular formation. DSC thermodynamic profiles indicate that the favorable folding of each hairpin results from the typical compensation of favorable enthalpy and unfavorable entropy contributions, while the DSC curves as a function of salt concentration yielded an uptake of cations and negative heat capacity effects. PPC melting curves yielded positive folding volumes ranging 12 to 31 cm3/mol, corresponding to releases of water molecules; in contrast, an uptake of water (ranging from 32 to 63 mol of H2O/mol) is observed from osmotic stress experiments using ethylene glycol as the osmolyte. Overall, the increase in the size of the variable bulge or internal loop yielded lower TMs and slightly more favorable enthalpies, corresponding to less favorable free energy contributions of ~0.7 kcal/mol per thymine residue. The volume measurements will be correlated with the unfolding entropies and discussed in terms of the type of water that is hydrating these stem-loop motifs structures.

This research has been supported by Grants MCB-0616005 and MCB-1122029 from the National Science Foundation.

Irine Khutshivilli
Iztok Prislan
Luis A. Marky

Department of Pharmaceutical Sciences
University of Nebraska Medical Center
986025 Nebraska Medical Center
Omaha, NE 68198-6025

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