Structure guided fluorescence labeling reveals a two-step binding mechanism of neomycin to its RNA aptamer

The ability of the cytidine analog Ç(m)(f) to act as a position specific reporter of RNA-dynamics was spectroscopically evaluated. Ç(m)(f)-labeled single- and double-stranded RNAs differ in their fluorescence lifetimes, quantum yields and anisotropies. These observables were also influenced by the nucleobases flanking Ç(m)(f). This conformation and position specificity allowed to investigate the binding dynamics and mechanism of neomycin to its aptamer N1 by independently incorporating Ç(m)(f) at four different positions within the aptamer. Remarkably fast binding kinetics of neomycin binding was observed with stopped-flow measurements, which could be satisfactorily explained with a two-step binding. Conformational selection was identified as the dominant mechanism.