title: An electrochemiluminescent aptamer switch for a high-throughput assay of an RNA editing reaction.
abstract: An RNA editing reaction that is both essential and specific to the trypanosomatid parasites is an attractive target for new drug development. Although high-throughput screening of chemical libraries is a powerful strategy often used to identify new drugs, the available in vitro editing assays do not have the necessary sensitivity and format for this approach to be feasible. A ruthenium labeled reporter RNA is described here that overcomes these limitations as it can both detect edited product in the low femtomole range and is ideal for high-throughput format. The reporter RNA consists of an RNA editing substrate linked to a streptavidin-binding aptamer that is initially held within an inactive conformation. An in vitro selection strategy optimized the linkage so that the streptavidin-binding aptamer is only activated by an editing-induced conformational change. An electrochemiluminescent signal results from the ruthenium label when the reporter is bound to the bottom of a streptavidin-coated microtiter plate where it can be stimulated by a carbon electrode. Chemical probing, mutagenesis, and binding affinity measurements were used to characterize the reporter. The highly sensitive assay could be adapted to a broad range of RNA processing reactions.
authors.affiliation: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
authors.forename: Shuang
authors.initials: S
authors.lastname: Liang
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Connell
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D004563:Electrochemistry; D049449:Luminescence; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D017393:RNA Editing; D012689:Sequence Homology, Nucleic Acid
chemical_list: D052157:Aptamers, Nucleotide
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