Book of Abstracts: Albany 2011

category image Albany 2011
Conversation 17
June 14-18 2011
©Adenine Press (2010)

Temperature-Induced Unfolding Thermodynamics of DNA Hairpins Containing Internal Loops and Their targeting with Complementary Strands

The main focus of our research is to further our understanding of the physico-chemical properties of nucleic acid structures. In particular, to investigate the melting behavior of unusual DNA structures, by determining their complete unfolding thermodynamic profiles. In this work, we use a set of DNA hairpins as a model to mimic a common motif present in the secondary structures of mRNA, i.e., a stem-loop motif with an internal loop in the stem. Specifically, we used a combination of UV spectroscopy and differential scanning calorimetry (DSC) melting techniques to determine the unfolding thermodynamics of a set of hairpins with sequence:

d(GCGCTnGTAACT5GTTACTnGCGC, where Tn corresponds to internal loops with n = 1, 3 or 5 and “T5” is an end loop of 5 thymines, as shown in the figure. The UV melting curves of each hairpin show monophasic transition with TMs that are independent of strand concentration, confirming their intramolecular formation. Analysis of the DSC profiles indicates that the unfolding of each hairpin results from the typical compensation of a unfavorable enthalpy (breaking of base-pair stacks) and favorable entropy contributions (release of ion and water molecules). The increase in the size of the internal loop from 2 to 10 thymines yielded: a) lower TMs and similar enthalpy contributions; b) lower heat capacity values that correlated with the lower releases of structural water molecules; and c) higher ion releases. Furthermore, we used isothermal titration calorimetry and DSC to investigate directly and indirectly, respectively, the thermodynamics of the reaction of each hairpin with their partially complementary strands. The results showed that all three targeting reactions yielded favorable free energy contributions that were enthalpy driven. Overall, this approach works because of the favorable heat contributions resulting from the formation of base-pair stacks involving the unpaired bases of the loops. Supported by Grant MCB-0315746 from the NSF.

Luis A. Marky
Iztok Prislan
Cynthia Lee
and Hui-Ting Lee

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