Similar conformations of hairpins with TTT and TTTT sequences: NMR and molecular modeling evidence for T.T base pairs in the TTTT hairpin.

The conformations of the d[G(1)C(2)G(3)C(4)-T(a)T(b)T(c)T(d)-G(5)C(6)G(7)C(8)] (T4) and d[G(1)C(2)G(3)C(4)-T(a)T(b)T(c)-G(5)C(6)G(7)C(8)] (T3) DNA hairpins have been studied. The 1H and 31P signals of the two hairpins have been nearly completely assigned by means of two-dimensional NMR spectroscopy in D2O (NOESY (two-dimensional nuclear Overhauser effect and exchange spectroscopy) at mixing times of 5, 50, 100, 300 and 500 ms, double-quantum-filtered correlation spectroscopy (DQF-COSY) and 1H-31P reverse chemical shift correlation (RCSC), and one-dimensional NOE spectra in 90% H2O. Conformational analysis using distance geometry (DG), molecular mechanics (MM) and molecular dynamics (MD) gave model conformations, which were evaluated by comparison of experimental and simulated 2D NOESY spectra. For the T4 sequence in T4, both NMR data and modeling indicated a T(a).T(d) wobble base pair. Although two types of T(a).T(d) base pairs are possible, the one with T(a)NH-T(d)O4 and T(a)O2-T(d)NH H-bonds was calculated to be more stable. Because the T(a).T(d) base pair of T4 extends the stem, there are only two residues (T(b) and T(c) in the loop. Although there are three residues in the T3 loop, the T(c) base projects into the solvent. The resulting conformational models have very similar loop folding patterns (FP): the bases of the two adjacent residues that begin the loop [T(b)T(c) of T4 and T(a)T(b) or T3] have a minor groove/major groove orientation with the first residue each having a trans alpha torsion angle; and the phosphodiester group that links the residues at the 3' end of the loop and the 5' top of the stem [T(c)pT(d) of T4 and T(c)pG(5) of T3] has a gauche+, gauche+ zeta,alpha conformation with a trans gamma angle for the second residue in both. These or similar features appear to be present in most of the few other hairpins studied previously by conformational methods. Thus, we believe that the conformations of the loops in T3 and T4 hairpins have greater similarities than previously recognized.