Aminoglycoside antibiotics can inhibit or activate twister ribozyme cleavage

The twister ribozyme is a widely distributed self‐cleaving RNA enzyme. The aminoglycoside antibiotics are a clinically important class of flexible RNA‐binding ligands. Aminoglycoside binding can modulate ribozyme activity such that paromomycin inhibits and sisomicin activates the ribozyme. Drug binding induces allosteric changes to the tertiary structure of the RNA to hinder or facilitate interactions at the active site of the ribozyme slowing or accelerating bond scission. Interactions between aminoglycoside antibiotics and the twister ribozyme were investigated in this study. An initial screen of 17 RNA‐binding antibiotics showed that a number of aminoglycosides inhibit the ribozyme, while a subset of aminoglycosides enhances twister cleavage. Initial kinetic analysis of the twister ribozyme showed a sevenfold inhibition of ribozyme cleavage by paromomycin and a fivefold enhancement of cleavage by sisomicin. Direct binding between the twister ribozyme RNA and paromomycin or sisomicin was measured by microscale thermophoresis. Selective 2′‐hydroxyl acylation analysed by primer extension shows that both paromomycin and sisomicin induce distinctive tertiary structure changes to the twister ribozyme. Published crystal structures and mechanistic analysis of the twister ribozyme have deduced a nucleobase‐mediated general acid–base catalytic mechanism, in which a conserved guanine plays a key role. Here, we show that paromomycin binding induces a structural transition to the twister ribozyme such that a highly conserved guanine in the active site becomes displaced, leading to inhibition of cleavage. In contrast, sisomicin binding appears to change interactions between P3 and L2, inducing allosteric changes to the active site that enhance twister RNA cleavage. Therefore, we show that small‐molecule binding can modulate twister ribozyme activity. These results suggest that aminoglycosides may be used as molecular tools to study this widely distributed ribozyme.