The duplex (rGAGGUCUC)2 contains the motif [sequence: see text] which is unusually stable compared with other symmetric tandem GU mismatches and occurs in the P5 helix of the group I intron of Tetrahymena thermophila. The three-dimensional solution structure of (rGAGGUCUC)2 was determined using two-dimensional NMR and a simulated annealing protocol. The structure is remarkably similar to the A-DNA crystal structure of (dGGGGTCCC)2 [Kneale, G., Brown, T., & Kennard, O. (1985) J. Mol. Biol. 186, 805-814] which contains the analogous motif [sequence: see text]. Incorporation of the [sequence: see text] motif has little effect on backbone torsion angles and helical parameters compared with standard A-form. The only significant departure from A-form is a slight overtwisting 5' of the G in the GU mismatch and a displacement of the mismatches toward the minor groove. Inspection of stacking patterns of this structure and comparison with symmetric tandem GT mismatches in A-DNA oligonucleotides from crystal structure data suggest that electrostatics are important in determining motif stability.

The stabilities and structures of a series of RNA octamers containing symmetric tandem mismatches were studied by UV melting and imino proton NMR. The free energy increments for tandem mismatch formation are found to depend upon both mismatch sequence and adjacent base pairs. The observed sequence dependence of tandem mismatch stability is UGGU > GUUG > GAAG > or = AGGA > UUUU > CAAC > or = CUUC approximately UCCU approximately CCCC approximately ACCA approximately AAAA, and the closing base pair dependence is 5'G3'C > 5'C3'G > 5'U3'A approximately 5'A3'U. These results differ from expectations based on models used in RNA folding algorithms and from the sequence dependence observed for folding of RNA hairpins. Imino proton NMR results indicate the sequence dependence is partially due to hydrogen bonding within mismatches.

The thermodynamic stabilities and structures of a series of RNA duplexes containing nonsymmetric tandem mismatches in the context of , where are tandem mismatches, were studied by UV melting and imino proton NMR. The contribution of one mismatch to the free energy increment for tandem mismatch formation depends on the identity of the other mismatch. Imino proton NMR indicates that this is partly because the structure of a mismatch is dependent on the adjacent mismatch. The results suggest that differences in size, shape, and hydrogen bonding of the adjacent mismatches play important roles in determining loop stability. A model for predicting stabilities of all possible tandem mismatches is proposed based on these and previous results.

G.U pairs occur frequently and have many important biological functions. The stability of symmetric tandem G.U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G.U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G.U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochemistry 34, 3204-3211]. In particular, at 37 degrees C, the motif 5'-CGUG-3' is less stable by approximately 3 kcal/mol compared with other symmetric tandem G.U motifs with G-C as adjacent pairs: 5'-GGUC-3', 5'-GUGC-3', and 5'-CUGG-3'. The solution structures of r(GAGUGCUC)(2) and r(GGCGUGCC)(2) duplexes have been determined by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G.U pair region. The striking discovery is that in r(GGCGUGCC)(2) each G.U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addition, the temperature dependence of the imino proton resonances is also consistent with the different number of hydrogen bonds in the G.U pair. To test the NMR models, U or G in various G.U pairs were individually replaced by N3-methyluridine or isoguanosine, respectively, thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.