Multiplexed screening of neutral mass-tagged RNA targets against ligand libraries with electrospray ionization FTICR MS: a paradigm for high-throughput 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 tag to one of the RNA targets shifts the detected peaks to a higher mass/charge ratio, where complexes with small molecules can be identified unambiguously. An appropriately placed neutral mass tag does not alter RNA--ligand binding. The utility of this strategy is demonstrated with model RNAs corresponding to the decoding region of the prokaryotic and eukaryotic rRNAs and a mixture of five aminoglycosides. Complexes are observed between the aminoglycoside library and the prokaryotic rRNA model, while no aminoglycoside was observed to bind to the mass-tagged eukaryotic rRNA model. The differential binding data is consistent with the eukaryotic A-site rRNA having a different conformation compared with the prokaryotic A-site that prevents entry and binding of neomycin-class aminoglycosides. Mass spectrometric analysis of neutral mass-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.
Hofstadler
S A
SA
Ibis Therapeutics, A Division of Isis Pharmaceuticals, Carlsbad, California 92008, USA.
Sannes-Lowery
K A
KA
Crooke
S T
ST
Ecker
D J
DJ
Sasmor
H
H
Manalili
S
S
Griffey
R H
RH
D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D009690:Nucleic Acid Conformation; D019151:Peptide Library; D012313:RNA / Q000737:chemistry; D012329:RNA, Bacterial / Q000737:chemistry; D012335:RNA, Ribosomal / Q000737:chemistry
D008024:Ligands; D019151:Peptide Library; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012313:RNA