Thermodynamics of d(GGGGCCCC) Binding to Neomycin-Class Aminoglycosides.

DNA adopts a number of conformations that can affect its binding to other macromolecules. The conformations (A, B, Z) can be sequence- and/or solution-dependent. While AT-rich DNA sequences generally adopt a Canonical B-form structure, GC-rich sequences are more promiscuous. Recognition of GC-rich nucleic acids by small molecules has been much more challenging than the recognition of AT-rich duplexes. Spectrophotometric and calorimetric techniques were used to characterize the binding of neomycin-class aminoglycosides to a GC-rich DNA duplex, GC, in various ionic and pH conditions. Our results reveal that binding enhances the thermal stability of GC, with thermal enhancement decreasing with increasing pH and/or Na concentration. Although GC bound to aminoglycosides demonstrated a mixed A- and B-form conformation, circular dichroism studies indicate that binding induces a conformational shift toward A-form DNA. Isothermal titration calorimetry studies reveal that aminoglycoside binding to GC is linked to the uptake of protons at pH = 7.0 and that this uptake is pH-dependent. Increased pH and/or Na concentration results in a decrease in GC affinity for the aminoglycosides. The binding affinities of the aminoglycosides follow the expected hierarchy: neomycin > paromomycin > ribostamycin. The salt dependence of DNA binding affinities of aminoglycosides is consistent with at least two drug NH groups participating in electrostatic interactions with GC. These studies further embellish our understanding of the many factors facilitating recognition of GC-rich DNA structures as guided by their optimum charge and shape complementarity for small-molecule amino sugars.