Aminoglycoside antibiotics drive RNA phase separation

<jats:p>Aminoglycoside antibiotics bind RNA with high affinity through networks of amine and hydroxyl contacts, yet whether this multivalent binding can drive macroscopic RNA phase transitions has never been tested. Here we show that aminoglycosides are a class of small-molecule RNA condensers, and we take neomycin B (neoB), an FDA-approved member of the family, as a representative drug through which to dissect the mechanism. NeoB induces concentration-dependent phase separation of poly(A), poly(U), and total \textit{E.~coli} RNA, and kanamycin, apramycin, and gentamicin condense RNA as well. Condensate size and density are tunable by pH and ionic strength, which independently modulate neoB protonation and screening of interdroplet repulsion. NeoB forms more stable condensates than spermine despite spermine's larger effective charge at physiological pH, whereas the amine-free polyol fucitol fails to condense RNA. Molecular dynamics simulations attribute neoB's greater efficiency to additional hydrogen bonds donated by its hydroxyl groups. The chemical complexity that aminoglycosides evolved for RNA recognition thus also drives a macroscopic RNA phase transition that may contribute to bactericidal activity and cellular toxicity.</jats:p>