Natural abundance heteronuclear NMR studies of the T-3 mini-loop hairpin in the terminal repeat of the adenoassociated virus 2

A DNA hairpin containing a T-3 loop, as occurs in the terminal repeat of a popular gene therapy vector (Adenoassociated Virus 2, AAV2), has been exten sively studied using homo- and heteronuclear NMR experiments. Almost comple te assignment of the proton and carbon resonances, including H5'((Pro-S)) a nd H5'((Pro-R)) protons, has been accomplished at natural abundance. NOESY spectra in H2O and D2O have revealed many unusual NOEs, which, when combine d with the epsilon, beta, gamma, and chi torsion angles determined from het eronuclear H-1-C-13, H-1- P-31, and C-13-P-31 coupling constants, have allo wed for a more detailed picture of the T-3 mini-loop hairpin. The three loo p 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. Co ntrary to an RNA oligomer with a central U-3 sequence that has the tendency to form a duplex with three U . U mismatches, the d(GAAGC-TTT-GCTTC) seque nce exists mostly as a hairpin under millimolar NMR conditions. Since T-3 t riloop was found to be an essential element for the site-specific non-homol ogous integration of the AAV2 virus, and modification of the T-3 loop resid ue abolishes such capability, the structure we report here may be of biolog ical significance.