Melting of a DNA Hairpin without Hyperchromism

Abstract

UV absorbance spectroscopy is the most common method for detecting nucleic acid structural transitions and obtaining thermodynamic parameters. UV-detected melting has been used to determine stabilities of nucleic acid hairpins, duplexes, triplexes, and higher order structures and to determine thermodynamic effects of unusual or modified bases and mismatched base-pairs. We report that in some cases UV absorbance spectroscopy is an inadequate analytical technique for these purposes. Some critical transitions are invisible to UV absorbance spectroscopy. For example, the conversion of dodecamer d(CGCAAATTCGCG) from hairpin to random coil is not accompanied by hyperchromism. Circular dichroism (CD) spectroscopy (263 nm) clearly detects two transitions for this dodecamer, each giving a pronounced change in ellipiticity. The concentration dependence of the low-temperature transition and the concentration independence of the high-temperature transition indicate that the predominant state converts from duplex to hairpin to random coil as the temperature increases. These assignments are confirmed by comparison to oligonucleotides of similar sequence that undergo a hairpin to coil transition only. In contrast to CD spectroscopy, UV absorbance spectroscopy shows only a single transition. The transition detected by UV absorbance spectroscopy corresponds to the low-temperature transition detected by CD. UV absorbance spectroscopy does not detect the second transition at any wavelength (from 218 to 310 nm) (by changes) in either absorbance or its derivative with temperature.