DIRECT OBSERVATION OF AMINOGLYCOSIDE-RNA INTERACTIONS BY SURFACE-PLASMON RESONANCE

The specificity of neomycin B and related aminoglycoside antibiotics i n their interaction with the Rev responsive element (RRE) of HIV-1 mRN A has been studied by directly observing the aminoglycoside-RNA comple xes using surface plasmon resonance. Several different RNA sequences, each with a biotin tag, have been prepared using T7 RNA polymerase-cat alyzed transcription of synthetic DNA templates and have been immobili zed on a streptavidin-coated surface for the binding study, The result s indicate that neomycin B is not specific for the G-rich bubble regio n in RRE. Rather, it appears to interact with three different sites, e ach with a submicromolar dissociation constant, within the 67-nucleoti de domain II of RRE. Further analysis of neomycin B binding with three short synthetic RNA hairpins showed binding with submicromolar affini ty and 1:1 stoichiometry in each case. This suggests that neomycin B m ay generally bind with this affinity to regular A-form RNA or hairpin loops. The approach described here is generally useful for understandi ng the fundamental interactions involved in the specific recognition o f nucleic acids by small molecules which is the basis of rational drug design.