Title:An Image-Based Biosensor Assay Strategy to Screen for Modulators of the microRNA 21 Biogenesis Pathway
Volume: 15
Issue: 7
Author(s): David Shum, Bhavneet Bhinder, Constantin Radu, Paul Calder, Christina N. Ramirez and Hakim Djaballah
Affiliation:
Keywords:
High throughput screening, image-based biosensor, miRNA biogenesis, modulator, EGFP, RNA molecules, Gene regulation, microarray, fluorescence signal output, Automation System
Abstract: microRNAs (miRNAs) are evolutionary conserved, small endogenous non-coding, RNA molecules. Although
their mode of action has been extensively studied, little is known about their biogenesis. As their altered expression has
been implicated in many diseases, small molecules that would modulate their expression are sought after. They are
generated through the concerted action of several complexes which promote their transcription, maturation, export,
trafficking, and loading of mature miRNA into silencing complexes. An increasing number of studies have suggested that
each of these steps serves as a regulatory junction in the process, and therefore provides an intervention point. For this
purpose, we have developed a simple image-based assay strategy to screen for such modulators. Here, we describe its
successful implementation which combines the use of a microRNA 21 (miR-21) synthetic mimic together with an EGFP
based reporter cell line, where its expression is under the control of miR-21, to monitor EGFP expression in a format
suitable for HTS. The strategy was further validated using a small panel of known gene modulators of the miRNA
pathway. A screen was performed in duplicate against a library of 6,912 compounds and identified 48 initial positives
exhibiting enhanced EGFP fluorescence intensity. 42 compounds were found to be inherently fluorescent in the green
channel leaving the remaining 6 as potential inhibitors and with a positive rate of 0.09%. Taken together, this validated
strategy offers the opportunity to discover novel and specific inhibitors of the pathway through the screening of diverse
chemical libraries.
