Structural Features and Stability of an RNA Triple Helix in Solution

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Abstract
A 30 nt RNA with a sequence designed to form an intramolecular triple helix was analyzed by one- and two-dimensional NMR spectroscopy and UV absorption measurements. NMR data show that the RNA contains seven pyrimidine-purine-pyrimidine base triples stabilized by Watson-Crick and Hoog-steen interactions. The temperature dependence of the imino proton resonances, as well as UV absorption data, indicate that the triple helix is highly stable at acidic pH, melting in a single sharp transition centered at 62°C at pH 4.3. The Watson-Crick and Hoogsteen pairings are disrupted simultaneously upon melting. The NMR data are consistent with a structural model where the Watson-Crick paired strands form an A-helix. Results of model building, guided by NMR data, suggest a possible hydrogen bond between the 2′ hydroxyl proton of the Hoogsteen strand and a phosphate oxygen of the purine strand. The structural model is discussed in terms of its ability to account for some of the differences in stability reported for RNA and DNA triple helices and provides insight into features that are likely to be important in the design of RNA binding compounds.