Biomolecules: Melting point and thermodynamics of DNA

The melting temperature of DNA refers to the temperature at which 50% of DNA in a sample has denatured from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA). Sensitive measurement of the melting curve of a sample of DNA can be used to detect single nucleotide differences between two DNA samples. This technique is possible because guanine-cytosine (GC) pairs contribute greater stability to dsDNA than adenosine-thymine (AT) pairs. Figure 1 shows three samples of DNA with different percentages of GC content.

Figure 1 - DNA melting curves for three strands of DNA with different levels of GC content. The y-axis indicates the fraction of DNA molecules that are single-stranded.

To better understand the nature of DNA melting, researchers characterized how different structural elements of dsDNA affect its stability and therefore its melting temperature. They suspected that the two main contributors to dsDNA stability would be hydrogen bonding between base pairs and pi-stacking, a non-covalent interaction that occurs only between the aromatic portions of bases. They were able to isolate the free energy contributions of individual structural elements by making chemical alterations that effectively eliminated the free energy contributions of all other structural elements. Table 1 shows the free energy contributions of the different structural elements they investigated. They also observed how DNA stability was affected by salt concentration, pH, and the presence of DNA intercalators, aromatic compounds that can be toxic or mutagenic due to their ability to insert between DNA bases. Table 2 shows how each of these factors changes the free energy (∆G) of dsDNA formation, represented by ∆∆G.

Table 1 - Free energy (∆G) contributions of individual non-covalent interactions to the thermodynamic stability of dsDNA. Experiments were performed in cellular conditions (T = 37 °C).

Table 2 - Changes to free energy (∆∆G) of dsDNA formation in various conditions. Experiments were performed in cellular conditions (T = 37 °C).