Investigating the Intramolecular Competition of Different RNA Binding Motifs for Neomycin B by Native Top‐Down Mass Spectrometry.

The ongoing search for small molecule drugs that target ribonucleic acids (RNA) is complicated by a limited understanding of the principles that govern RNA‐small molecule interactions. Here we have used stoichiometry‐resolved native top‐down mass spectrometry (MS) to study the binding of neomycin B to small model hairpin RNAs, an unstructured RNA, and a viral RNA construct. For 15–22 nt model RNAs with hairpin structure, we found that neomycin B binding to hairpin loops relies on interactions with both the nucleobases and the 2′‐OH groups, and that a simple 5′ or 3′ overhang can introduce an additional binding motif. For a 47 nt RNA construct derived from stem IA of the human immunodeficiency virus 1 (HIV‐1) rev response element (RRE) RNA, native top‐down MS identified four different binding motifs, of which the purine‐rich internal loop showed the highest affinity for neomycin B. Stoichiometry‐resolved binding site mapping by native top‐down MS allows for a new perspective on binding specificity, and has the potential to reveal unexpected principles of small molecule binding to RNA. [ABSTRACT FROM AUTHOR]