Natural abundance heteronuclear NMR studies of the T3 mini-loop hairpin in the terminal repeat of the adenoassociated virus 2

A DNA hairpin containing a T3 loop, as occurs in the terminal repeat of a popular gene therapy vector (Adenoassociated Virus 2, AAV2), has been extensively studied using homo- and heteronuclear NMR experiments. Almost complete assignment of the proton and carbon resonances, including H5'(Pro-S) and H5'(Pro-R) protons, has been accomplished at natural abundance. NOESY spectra in H2O and D2O have revealed many unusual NOEs, which, when combined with the ε, β, γ, and χ torsion angles determined from heteronuclear 1H-13C, 1H-31P, and 13C-31P coupling constants, have allowed for a more detailed picture of the T3 mini-loop hairpin. The three loop thymidines are all unpaired, yet are highly structured when bracketed by a 5'-GC...GC-3' stem sequence. The structure determined in this manuscript is considerably different from several other structures reported so far. Contrary to an RNA oligomer with a central U3 sequence that has the tendency to form a duplex with three U.U mismatches, the d(GAAGC-TTT-GCTTC) sequence exists mostly as a hairpin under millimolar NMR conditions. Since T3 triloop was found to be an essential element for the site-specific non-homologous integration of the AAV2 virus, and modification of the T3 loop residue abolishes such capability, the structure we report here may be of biological significance