Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry

We have developed methods for studying the interactions between small molec ules and RNA and have applied them to characterize the binding of three cla sses of aminoglycoside antibiotics to ribosomal RNA subdomains. High-resolu tion MS was used to quantitatively identify the noncovalent binding interac tions between mixtures of aminoglycosides and multiple RNA targets simultan eously. Signal overlap among RNA targets was avoided by the addition of neu tral mass tags that direct each RNA target to a unique region of the spectr um. In addition to determining binding affinities, the locations of the bin ding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycoside/RNA complex. Specific complexes were obse rved for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomyc in, and lividomycin, whereas apramycin preferentially formed a complex with the eukaryotic subdomain. We show that differences in binding between paro momycin and ribostamycin can be probed by using an MS-MS protection assay. We have introduced specific base substitutions in the RNA models and have m easured their impact on binding affinity and selectivity. The binding of ap ramycin to the prokaryotic subdomain strongly depends on the identity of po sition 1408, as evidenced by the selective increase in affinity for an A140 8G mutant. An A1409-G1491 mismatch pair in the prokaryotic subdomain enhanc ed the binding of tobramycin and bekanamycin. These observations demonstrat e the power of MS-based methods to provide molecular insights into small mo lecule/RNA interactions useful in the design of selective new antimicrobial drugs.