The binding of neomycin B and related aminoglycoside antibiotics to the prokaryotic ribosomal RNA decoding region has been investigated using a recently developed surface plasmon resonance assay. A number of naturally occurring aminoglycosides containing a neamine or neamine-like substructure bind specifically to a model of the site of the ribosomal decoding region RNA. This recognition event is the basis of the antibacterial activity of this class of compounds. A series of analogs was designed and synthesized to probe the role of neomycin ring IV (2,6-dideoxy-2,6-diamino-beta-L-idopyrano The binding results indicate that the positive charge presented on the idose ring is necessary for specific binding in vitro and cannot be replaced by amines attached via flexible linkers. However, the antibiotic activity (minimum inhibitory concentration) of the analog where ring IV is replaced with st diamine tail is the same as neomycin B in a liquid culture assay against Escherichia coli.