Document Detail


Microfluidic screening of electrophoretic mobility shifts elucidates riboswitch binding function.
MedLine Citation:
PMID:  23343213     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 conformational change. In 0.3% of the time required for benchtop assays (3.2 versus 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 (K(d)) for both rapidly and slowly interconverting riboswitch-ligand pairs as validated through experiments and modeling. Conformational change, triplicate mobility shift measurements, and K(d) 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.
Authors:
Kelly Karns; Jacob M Vogan; Qian Qin; Scott F Hickey; Stephen C Wilson; Ming C Hammond; Amy E Herr
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2013-02-11
Journal Detail:
Title:  Journal of the American Chemical Society     Volume:  135     ISSN:  1520-5126     ISO Abbreviation:  J. Am. Chem. Soc.     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-02-27     Completed Date:  2013-08-12     Revised Date:  2014-03-19    
Medline Journal Info:
Nlm Unique ID:  7503056     Medline TA:  J Am Chem Soc     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3136-43     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Electrophoretic Mobility Shift Assay / methods*
Microfluidics / methods*
RNA / chemistry*
Riboswitch*
Grant Support
ID/Acronym/Agency:
1DP2OD007294/OD/NIH HHS; 1DP2OD008677/OD/NIH HHS; DP2 OD007294/OD/NIH HHS; DP2 OD008677/OD/NIH HHS; T32 GM007232/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Riboswitch; 63231-63-0/RNA
Comments/Corrections

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