Microfluidic Screening of Electrophoretic Mobility Shifts Elucidates Riboswitch Binding Function

Journal of the American Chemical Society (Impact Factor: 12.11). 01/2013; 135(8). DOI: 10.1021/ja310742m
Source: PubMed


Riboswitches are RNA sensors that change conformation upon binding small molecule metabolites, in turn modulating gene expression. Our understanding of riboswitch regulatory function would be accelerated by a high throughput, quantitative screening tool capable of measuring riboswitch-ligand binding. We introduce a microfluidic mobility shift assay that enables precise and rapid quantitation of ligand binding and subsequent riboswitch conforma-tional change. In 0.3% of the time required for bench top assays (3.2 min vs. 1020 min), we screen and validate five candidate SAM-I riboswitches isolated from thermophilic and cryophilic bacteria. The format offers enhanced resolution of conformational change compared to slab gel formats, quantitation and repeatability for statistical assessment of small mobility shifts, low reagent consumption, and riboswitch characterization without modification of the aptamer structure. Appreciable analytical sensitivity coupled with high resolution separation performance allows quantitation of equilibrium dissociation constants (Kd) for both rapidly and slowly interconverting riboswitch-ligand pairs as validated through experiments and modeling. Conformational change, triplicate mobility shift measurements, and Kd are reported for both a known and a candidate SAM-I riboswitch with comparison to in-line probing assay results. The microfluidic mobility shift assay establishes a scalable format for the study of riboswitch-ligand binding that will advance the discovery and selection of novel riboswitches and the development of antibiotics to target bacterial riboswitches.

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    • "Metabolites were quantified by liquid chromatography-mass spectrometry analysis (LC-MS), as described previously (Mayfield et al. 2012). For detection of SAM by the Pi SAM-I riboswitch aptamer, extracts were diluted 1:50, and 1 ml was applied to 9 ml radiolabeled RNA in TBMK buffer (90 mM Tris, 89 mM boric acid, 10 mM MgCl 2 , 10 mM KCl), as described previously (Karns et al. 2013). Before mixing with lysate, the RNA had been renatured at 70° for 3 min and then allowed to return to room temperature for 10 min. "
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