Aminoglycoside antibiotics drive RNA phase separation

AbstractAminoglycoside 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 neomycin B (neoB), an FDA-approved aminoglycoside, induces concentration-dependent phase separation of poly(A), poly(U), and total E. coli RNA. Condensate size and density are tunable by pH and ionic strength, which modulate neoB protonation and screening of interdroplet repulsion independently. Comparison with spermine shows that neoB produces more stable condensates despite spermine carrying at least as much effective charge at physiological pH. Fucitol, a hydroxyl-rich polyol lacking amines, fails to condense RNA, showing that cationic amine groups are required and cannot be replaced by hydroxyl groups. Molecular dynamics simulations attribute the greater condensation efficiency of neoB to additional hydrogen bonds donated by its hydroxyl groups. These results reveal that the chemical complexity of aminoglycosides, evolved for tight RNA recognition, also drives a macroscopic RNA phase transition. RNA condensation may contribute to aminoglycoside bactericidal activity and cellular toxicity.