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

Abstract

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.