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.
As part of a search for organic compounds that selectively target RNA, we found that specific diphenylfuran derivatives, which are related to compounds that bind to the DNA minor groove, bind very strongly to RNA in a manner very sensitive to the structure of the compounds. In extended development of the diphenylfuran series, we found that a tetracationic heterocycle containing a phenyl-furan-benzimidazole unfused aromatic system, DB340, exhibits pronounced selectivity for the RRE RNA stem-loop from HIV-1. We report here RNA footprinting, spectroscopic analysis, affinity determinations, and initial NMR structural results of the complex. The results indicate that DB340 binds to RRE in a highly structured and cooperative complex at a 2:1 DB340 to RRE ratio. Overlap in the NMR spectra prevents detailed description of binding interactions at this time, but we are able to place DB340 in the RNA minor groove. Additionally, footprinting results and studies with mutant RRE sequences indicate that the internal loop of RRE is required for specific binding of DB340 as with the Rev protein. These results provide exciting new ideas for rational drug design with RNA as is now common with DNA and proteins.
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