Direct Observation of Aminoglycoside–RNA Binding by Localized Surface Plasmon Resonance Spectroscopy

Abstract

RNA is involved in fundamental biological functions when bacterial pathogens replicate. Identifying and studying small molecules that can interact with bacterial RNA and interrupt cellular activities is a promising path for drug design. Aminoglycoside (AMG) antibiotics, prominent natural products that recognize RNA specifically, exert their biological functions by binding to prokaryotic ribosomal RNA and interfering with protein translation, ultimately resulting in bacterial cell death. The decoding site, a small internal loop within the 16S rRNA, is the molecular target for the AMG antibiotics. The specificity of neomycin B, a highly potent AMG antibiotic, to the ribosomal decoding RNA site, was previously studied by observing AMG–RNA complexes in solution. Here we study this interaction using localized surface plasmon resonance (LSPR) transducers comprising gold island films prepared by evaporation on glass and annealing. Small molecule AMG receptors were immobilized on the Au islands via PEG-thiol linkers, and the interaction with target RNA in solution was studied by monitoring the change in the LSPR optical response upon binding. The results show high-affinity binding of neomycin to 27-nucleotide model A-site and A-modified RNA sequences in the nanomolar range, while no specific binding is observed for synthetic RNA oligomers (e.g., poly-U). The impact of specific base substitutions in the A-site RNA constructs on binding affinity and selectivity is determined quantitatively. It is concluded that LSPR is a powerful tool for providing molecular insight into small molecule–RNA interactions and for the design and screening of selective antimicrobial drugs.

Tip-Specific Functionalization of Gold Nanorods for Plasmonic Biosensing: Effect of Linker Chain Length

“…Most important, the functionalization with receptors imparts specificity by only binding the target analyte with high affinity. In this respect, many different types of biomolecular interactions have been explored using receptors based on antibodies, [4][5][6][7] peptides, 8 aptamers, 9,10 carbohydrates, 11,12 or nucleic acids. 13,14 Colloidal gold particles have interesting optical properties that have been explored in label-free detection schemes.…”

Understanding the Contributions of Conformational Changes, Thermodynamics, and Kinetics of RNA–Small Molecule Interactions

“…Tor, Vaskevich, Rubinstein, and co-workers analyzed the interaction of neomycin B with the 16S bacterial ribosomal decoding A site, human ribosomal 18S RNA, and two other constructs using localized SPR (Figures 5b and 7c). 115 Neomycin B had a 10-fold faster k on for bacterial A-site RNA than for human 18S RNA. Authors suggested that this difference in k on might be the basis for preferential binding of neomycin to the bacterial A site over the eukaryotic A site.…”

Strategies to Overcome the Action of Aminoglycoside-Modifying Enzymes for Treating Resistant Bacterial Infections

“…HTS using methods other than fluorescence-based probes, namely MS [125,126], NMR [127] and surface plasmon resonance [128] have also been developed to measure binding affinity for the RNA A-site. Isis Pharmaceuticals has developed a MS assay, which, in addition to determining binding affinities of AGs for RNAs, also identified binding specificity based on the fragmentation pattern of AG/RNA complexes [126].…”