Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Temperature-induced Unfolding of Unusual DNA Structures: Correlation of Optical and DSC Melting Curves with Fluorescence Melts Using 2-Aminopurine.
One focus of our research is to investigate the melting behavior of unusual DNA structures and to determine their unfolding thermodynamic profiles. In this work, we used a combination of UV, CD and fluorescence spectroscopies, and differential scanning calorimetric (DSC) techniques to investigate the temperature unfolding of a variety of DNA structures. The main objectives were to correlate optical and calorimetric melting curves with fluorescence melts obtained by observing the fluorescence changes of 2-aminopurine (2-AP) when incorporated into DNA, and to determine the specific thermodynamic contributions for the single incorporation of 2-AP. Specifically, we have investigated the following: a) a dodecamer duplex, 5?-CGCGAXTTCCGG/ 5?-CCGGAATTCGCG; b) a hairpin, 5?-GTXACGCAAGTTAC, ?GCAA? is the loop; c) an intramolecular pyrimidine triplex, 5?-A3XA3C5T7C5T7; and d) a G-quadruplex, 5?-G2T2G2TXTG2T2G2; where ?X? is 2-AP.
The UV, CD, Fluorescence and DSC melting curves for each of these four molecules show monophasic transitions with similar transition temperatures, TMs, and van?t Hoff enthalpies. This indicates that the fluorescence changes for the unstacking of 2-AP follow the unfolding of the whole molecule. Comparison of the DSC thermodynamic profiles of each 2-AP modified molecule with its corresponding unmodified oligonucleotide shows that the single placement of 2-AP is destabilizing; the differential free energy term, ΔΔG°, ranged from 2.0 kcal (duplex) to 3.1 kcal (hairpin), due to lower TMs of 3.9-8.1 °C and lower formation enthalpies, 5 kcal/mol (duplex) to 14.6 kcal/mol (hairpin). The one exception is the G-quadruplex that was stabilized with the incorporation of 2-AP (ΔΔG° of -0.9 kcal/mol), and due to a higher TM (by 6.1 °C) and more favorable enthalpy contribution of -4 kcal/mol.
The overall results indicate that on appropriately placed 2-AP can be used as a probe to monitor the temperature unfolding of a nucleic acid molecule. Furthermore, the destabilizing effect for the incorporation of a 2-AP-dT base pair between two dA-dT base pairs is due to lower stacking contributions; while the stabilizing effect of the TXT loop of the G-quadruplex is due to additional stacking contributions of this loop with the G-quartet at the top of this molecule. Supported by Grant MCB-0616005 from NSF.
Dept of Pharmaceutical Sciences