Poster abstracts
Poster number 28 submitted by Sydney Rosenblum
Detecting miRNA-miRBP interactions with a cell-based split-enzyme assay
Sydney Rosenblum (Program in Chemical Biology, University of Michigan), Daniel A. Lorenz (Program in Chemical Biology, University of Michigan), Amanda L. Garner (Medicinal Chemistry, University of Michigan)
Abstract:
Greater than one-thousand human miRNAs are believed to control the translation of greater than 60% of all protein-coding genes. Accordingly, miRNA dysregulation is a common hallmark of countless human diseases, including cancers, neurological diseases, and cardiac diseases, making miRNA and miRNA binding proteins (miRBPs) attractive drug targets. Recent advances in high-throughput technology have enabled large-scale screening of small molecules and natural products against RNA targets. However, subsequent characterization of promising chemical scaffolds is hindered by the lack of reliable assays designed to detect target engagement of RNA-targeted compounds or protein-RNA interaction inhibitors. Most existing methods of determining target engagement successfully measure ligand engagement of protein targets, but such systems cannot be easily adapted to detect RNA target engagement or the disruption of protein-RNA interactions. The development of novel methods of detecting RNA-ligand and RNA-protein interactions in cells will not only provide tools for studying miRNA-miRBP interactions, but will also facilitate the discovery of miRNA-targeted ligands and miRNA-miRBP inhibitors. Here I describe a cellular assay that utilizes the split-enzyme NanoBiT® (Promega) system to detect the miR-miRBP interaction between the pre-let7 class of miRNA and Lin28. This strategy is currently being expanded to enable the detection and study of various miR-miRBP interactions as well as measure the cellular activity of small molecule inhibitors of such interactions.
Keywords: microRNA , microRNA binding protein, assay