Multiplexed screening of neutral mass-tagged RNA targets against ligand libraries with electrospray ionization FTICR MS: A paradigm for high-throughout affinity screening

We demonstrate that binding of mixtures of aminoglycosides can be measured simultaneously against multiple RNA targets of identical length and similar (or identical) molecular weight. Addition of a neutral mass bg to one of t he RNA targets shifts the detected peaks to a higher mass/charge ratio, whe re complexes with small molecules can be identified unambiguously. An appro priately placed neutral mass tag does not alter RNA-ligand binding. The uti lity of this strategy is demonstrated with model RNAs corresponding to the decoding region of the prokaryotic and eukaryotic rRNAs and a mixture of fi ve aminoglycosides. complexes are observed between the aminoglycoside libra ry and the prokaryotic rRNA model, while no aminoglycoside was observed to bind to the mass-tagged eukaryotic rRNA model. The differential binding dat a is consistent with the eukaryotic A-site rRNA having a different conforma tion compared with the prokaryotic A-site that prevents entry and binding o f neomycin-class aminoglycosides. Mass spectrometric analysis of neutral ma ss-tagged macromolecular targets represents a new high-throughput screening paradigm in which the interaction of multiple targets against a collection of small molecules can be evaluated in parallel.