An electrochemiluminescent aptamer switch for a high-throughput assay of an RNA editing reaction

Shuang Liang, Gregory J Connell

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1929-1938
Number of pages10
JournalRNA
Volume15
Issue number10
DOIs
StatePublished - Oct 2009

Keywords

  • Electrochemiluminescence
  • High-throughput
  • Leishmania
  • RNA editing
  • Trypanosomatid

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