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.

Identification of specific inhibitors for a trypanosomatid RNA editing reaction

“…1A;Liang and Connell 2009). The reporter for the assay contains a streptavidin-binding aptamer that is activated by a conformational change in response to editing.…”

“…The assay reactions contained 100 fmol of reporter RNA and 60 units of an L. tarentolae editing extract that were prepared as previously described (Liang and Connell 2009); one unit of editing activity is defined as the amount that results in 1 fmol of correctly edited product in 1 h using our standard reaction conditions. The pre-edited reporter RNA was initially denatured for 5 min at 65°C in 2.4 volumes of denaturing buffer containing 0.2 mM EDTA and 25 mM Tris (pH 8.0, 27°C).…”

“…However, it is limited by the availability of crystal structures and by differences in structure or drug accessibility that can result from the study of individual proteins outside the context of the intact multi-protein complex. High-throughput screening (HTS) of large chemical libraries is an alternative strategy to identify inhibitors, but it only recently became feasible with the development of an assay with the required fidelity, sensitivity, and format (Liang and Connell 2009). The novel assay exploits an electrochemiluminescent (ECL) aptamer switch, and we describe here its application in an HTS of a chemical library.…”

Selection of binding targets in parasites using phage-display and aptamer libraries in vivo and in vitro

“…Among them, a RNA aptamer-based methodology was developed using a mitochondrial population from L. tarentolae as an initial source (Liang and Connell, 2009). The method detects the editing activity by an electrochemiluminescent signal generated by an editing responsive conformational change within a ruthenium labeled RNA reporter.…”

Applications of Electrochemiluminescence

“…Besides for the detection of proteins and small biomolecules, the combination of ECL and aptamers has also been used for a high-throughput assay of an RNA editing reaction [163]. RNA editing is the molecular process in which the information content in an RNA molecule is altered through a chemical change in the base makeup [164].…”