IMPROVED RNA STRUCTURE DETERMINATION BY DETECTION OF NOE CONTACTS TO EXCHANGE-BROADENED AMINO PROTONS

A 2D N-15-correlated NOESY experiment is described which makes it poss ible to obtain distance information for exchange-broadened amino proto ns in nucleic acids. Little or no NOE distance information is normally obtained for guanine and adenine amino protons because of rotation of the amino group around the C-N bond which leads to extreme broadening of the amino proton resonances. Here NOEs involving amino protons are detected via efficient transfer of heteronuclear magnetization betwee n the amino protons and nitrogens, which is accomplished by applicatio n of a train of closely spaced 180 degrees echo pulses. This technique has been applied to a uniformly N-15-labeled lead-dependent ribozyme known-as the leadzyme. The 2D N-15-correlated NOESY experiment allowed 19 NOE constraints to amino protons to be identified that had not bee n previously observed in standard NOE spectra. Molecular dynamics calc ulations were performed with and without these additional amino NOE co nstraints and demonstrated that these amino constraints significantly improved the structure of the GAAA tetraloop in the leadzyme. Such NOE constraints involving amino protons will likely improve structure det erminations of many other nucleic acid motifs.