Thermodynamics and structures of mismatched basepairs in RNA

Nuclear magnetic resonance (NMR) spectroscopy was employed in determining the solution structure of two RNA duplexes containing internal, tandem U-C mismatched basepairs. It was shown in both cases that the mispairs are stacked within the duplexes and form U-C base oppositions. However, the standard suite of NMR experiments employed were insufficient to select between different possible hydrogen-bonding structures for the mispairs. It is expected that utilization of more sophisticated NMR techniques such as isotropic labeling will help to better define RNA structure in solution.; Structure modeling of the RNA sequences, with the aid of NMR-derived restraints, produced well-defined structures for these mispairs differing for the two molecules. One exhibited a U-C mismatch opened towards the minor groove with only one direct hydrogen-bond. Such a conformation is capable of accommodating a water molecule in this groove to provide further stability through additional hydrogen-bonding. This is in agreement with an X-ray structure obtained for another RNA molecule with a U-C mismatch in a similar sequence contest.; Modeling of the second molecule showed structures with two direct hydrogen-bonds. One of the bonds, however, requires protonation of the cytosine N3. This conformation agrees with the observation of pH dependence in the NMR spectra seen in another RNA molecule with a U-C mismatch in the same sequence context. Additional resonances were seen as the pH was lowered below 5.5 indicating hydrogen-bonding involving a protonated base.; Thermodynamic measurements of RNA duplexes containing single mismatches showed that the penalty is mismatch dependent. The position of the mismatch was also seen to have a significant effect on stability with mismatches closer to the end of duplex regions being more stable. Refinement of the stability of mismatches in duplex RNA as a function of mismatch type and position should have a significant effect on thermodynamic predictions of RNA secondary structure.