Nuclear magnetic resonance spectroscopy and molecular modeling reveal thatdifferent hydrogen bonding patterns are possible for G center dot U pairs:One hydrogen bond for each G center dot U pair in r(GGC(GU)under-bar-GCC)(2) and two for each G center dot U pair in r(GAG(UG)under-bar-CUC)(2)

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 pa irs, i.e., 5'-<(UG)over bar>-3' > 5'-<(GU)over bar>-3', where an underline represents a nucleotide in a G . U pair [Wu, M., McDowell, J. A., and Turne r, D. H. (1995) Biochemistry 34, 3204-3211]. In particular, at 37 degrees C , the motif 5'-C<(GU)over bar>G-3' is less stable by approximately 3 kcal/m ol compared with other symmetric tandem G . U motifs with G-C as adjacent p airs: 5'-G<(GU)over bar>C-3', 5'-G<(UG)over bar>C-3', and 5'-C<(UG)over bar >G-3'. The solution structures of r(GAG<(UG)over bar>CUC)(2) and r(GGC<(GU) over bar>GCC)(2) duplexes have been determined by NMR and restrained simula ted annealing. The global geometry of both duplexes is close to A-form, wit h some distortions localized in the tandem G . U pair region. The striking discovery is that in r(GGC<(GU)over bar>GCC)(2) each G . U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobb le pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 i s 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 var ious G . U pairs were individually replaced by N3-methyluridine or isoguano sine, respectively, thus eliminating the possibility of hydrogen bonding be tween GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.