Aminoglycoside binding in the major groove of duplex RNA: The thermodynamic and electrostatic forces that govern recognition

We use a combination of spectroscopic, calorimetric, viscometric and comput er modeling techniques to characterize the binding of the aminoglycoside an tibiotic, tobramycin, to the polymeric RNA duplex, poly(rI).poly(rC), which exhibits the characteristic A-type conformation that is conserved among na tural and synthetic double-helical RNA sequences. Our results reveal the fo llowing significant features: (i) CD-detected binding of tobramycin to poly (rI)poly(rC) reveals an apparent site size of four base-pairs per bound dru g molecule; (ii) tobramycin binding enhances the thermal stability of the h ost poly(rI) poly(rC) duplex, the extent of which decreases upon increasing in Naf concentration and/or pH conditions; (iii) the enthalpy of tobramyci n-poly(rI) poly(rC) complexation increases with increasing pH conditions, a n observation consistent with binding-induced protonation of one or more dr ug amino groups; (iv) the affinity of tobramycin for poly(rI)poly(rC) is se nsitive to both pH and Na+ concentration, with increases in pH and/or Na+ c oncentration resulting in a concomitant reduction in binding affinity. The salt dependence of the tobramycin binding affinity reveals that the drug bi nds to the host RNA duplex as trication. (v) The thermodynamic driving forc e for tobramycin-poly(rI) poly(rC) complexation depends on pH conditions. S pecifically, at pH less than or equal to 6.0, tobramycin binding is entropy driven, but is enthalpy driven at pH > 6.0. (vi) Viscometric data reveal n on-intercalative binding properties when tobramycin complexes with poly(rI) poly(rC), consistent with a major groove-directed mode of binding. These d ata also are consistent with a binding-induced reduction in the apparent mo lecular length of the host RNA duplex. (vii) Computer modeling studies reve al a tobramycin-poly(rI) poly(rC) complex in which the drug fits snugly at the base of the RNA major groove and is stabilized, at least in part, by an array of hydrogen bonding interactions with both base and backbone atoms o f the host RNA. These studies also demonstrate an inability of tobramycin t o form a stable low-energy complex with the minor groove of the poly(rI) po ly(rC) duplex. In the aggregate, our results suggest that tobramycin-RNA re cognition is dictated and controlled by a broad range of factors that inclu de electrostatic interactions, hydrogen bonding interactions, drug protonat ion reactions, and binding-induced alterations in the structure of the host RNA. These modulatory effects on tobramycin-RNA complexation are discussed in terms of their potential importance for the selective recognition of sp ecific RNA structural motifs, such as asymmetric internal loops or hairpin loop-stem junctions, by aminoglycoside antibiotics and their derivatives. ( C) 2000 Academic Press.