MELTING OF A DNA HAIRPIN WITHOUT HYPERCHROMISM

UV absorbance spectroscopy is the most common method for detecting nuc leic acid structural transitions and obtaining thermodynamic parameter s. UV-detected melting has been used to determine stabilities of nucle ic acid hairpins, duplexes, triplexes, and higher order structures and to determine thermodynamic effects of unusual or modified bases and m ismatched base-pairs. We report that in some cases UV absorbance spect roscopy 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 (C D) spectroscopy (263 nm) clearly detects two transitions for this dode camer, each giving a pronounced change in ellipiticity. The concentrat ion dependence of the low-temperature transition and the concentration independence of the high-temperature transition indicate that the pre dominant state converts from duplex to hairpin to random coil as the t emperature increases. These assignments are confirmed by comparison to oligonucleotides of similar sequence that undergo a hairpin to coil t ransition only. In contrast to CD spectroscopy, UV absorbance spectros copy shows only a single transition. The transition detected by UV abs orbance spectroscopy corresponds to the low-temperature transition det ected by CD. UV absorbance spectroscopy does not detect the second tra nsition at any wavelength (from 218 to 310 nm) (by changes) in either absorbance or its derivative with temperature.