The RNA‐Bound Conformation of Neamine as Determined by Transferred NOE Experiments

Abstract

The tRNA(Phe)-bound conformation of the aminoglycoside neamine, a member of the neomycin B family, has been investigated by transferred NOE experiments in aqueous solution. This is the first time that the bioactive conformation of an RNA-bound aminoglycoside has been determined by this method. In buffers without divalent Mg(2+) ions, a high degree of electrostatically driven unspecific binding of aminoglycosides to the RNA was observed. Careful optimization of experimental conditions yielded buffer conditions optimized for cryo-probe NMR experiments. In particular, addition of Mg(2+) ions to the solutions was necessary to reduce the amount of unspecific binding as monitored by one-dimensional NMR and surface plasmon resonance experiments. CD spectroscopy was used to probe the effect of aminoglycosides and buffer conditions on the double helical content of tRNA(Phe). Finally the tRNA(Phe)-bound conformation of neamine was determined by trNOE build-up curves and compared with the previously reported crystal structure of neomycin B complexed to this RNA. Although the aminoglycoside in the crystal structure contains several configurational errors, the overall shape of the crystallographically determined RNA-bound structure is identical to the RNA-bound conformation defined by the NMR experiments. Therefore, the crystal structure has been refined by trNOE data. This is particularly important in the context of aminoglycosides being discussed as lead structures for the development of new anti-RNA drugs.