SOLUTION STRUCTURES OF 5-FLUOROURACIL-SUBSTITUTED RNA DUPLEXES CONTAINING G-U WOBBLE BASE-PAIRS

The structures and stabilities of three RNA duplexes that differed onl y in the position of 5-fluorouridine (FUrd) substitution were elucidat ed using NMR spectroscopy and UV hyperchromicity studies to determine if FUrd substitution altered the structure or stability of RNA duplexe s that contained G-U base pairs. The duplexes investigated corresponde d to the region of the U4-U6 snRNA complex that contained the 5' termi nus of U4 snRNA. The control duplex contained a G-U wobble base pair a nd also a G-A mismatched base pair. FUrd was substituted in one duplex at the G-U wobble base pair and in the second duplex at an A-U base p air adjacent to the wobble base pair. FUrd substitution slightly desta bilized the duplex that contained a G-FU base pair but stabilized the duplex that contained an A-FU base pair. NOESY spectra were used to de termine interproton distances, and these distance constraints were use d in a restrained molecular dynamics protocol to determine the three-d imensional structures of these RNA duplexes. Analyses of helical param eters, backbone torsion angles, and rms deviations between the final s tructures revealed no systematic differences due to FUrd substitution in RNA duplexes that contained G-U base pairs. The G-FU base pair adop ted wobble geometry, while the G-A mismatch formed a sheared base pair . NOESY spectra in H2O solution revealed the imino H-1 from FUrd excha nged more rapidly with solvent than did the Urd imino H-1 but did not show the G-FU base pair adopted an ionized structure. Reduced stacking occurred for the G-FU base pair relative to the G-U base pair in the time-averaged structure, and this, rather than ionization of the base pair, was responsible for the slight destabilization of the duplex tha t contained the G-FU base pair.