Native Top-Down Mass Spectrometry Uncovers Two Distinct Binding Motifs of a Functional Neomycin-Sensing Riboswitch Aptamer

Understanding how ligands bind to ribonucleic acids (RNA)is importantfor understanding RNA recognition in biological processes and drugdevelopment. Here, we have studied neomycin B binding to neomycin-sensingriboswitch aptamer constructs by native top-down mass spectrometry(MS) using electrospray ionization (ESI) and collisionally activateddissociation (CAD). Our MS data for a 27 nt aptamer construct revealthe binding site and ligand interactions, in excellent agreement withthe structure derived from nuclear magnetic resonance (NMR) studies.Strikingly, for an extended 40 nt aptamer construct, which representsthe sequence with the highest regulatory factor for riboswitch function,we identified two binding motifs for neomycin B binding, one correspondingto the bulge-loop motif of the 27 nt construct and the other one inthe minor groove of the lower stem, which according to the MS dataare equally populated. By replacing a noncanonical with a canonicalbase pair in the lower stem of the 40 nt aptamer, we can reduce bindingto the minor groove motif from & SIM;50 to & SIM;30%. Conversely,the introduction of a CUG/CUG motif in the lower stem shifts the bindingequilibrium in favor of minor groove binding. The MS data reveal site-specificand stoichiometry-resolved information on aminoglycoside binding toRNA that is not directly accessible by other methods and underscorethe role of noncanonical base pairs in RNA recognition by aminoglycosides.

Automated summary

Understanding how ligands bind to ribonucleic acids (RNA) is crucial in studying RNA recognition and drug development. This study used mass spectrometry (MS) to investigate neomycin B binding to neomycin-sensing riboswitch aptamer constructs. The MS data revealed the binding site and ligand interactions, providing valuable insights for RNA recognition by aminoglycosides.