Facilitating the Discovery of Chemical Tools for Manipulating pre-miRNA Structure-Function

Abstract

The biological implications of RNA continue to expand beyond the role as the simple messenger between DNA and protein translation. This expansion has been catalyzed by the development of new technologies that facilitate both the discovery of new RNAs and their biological impact. One class of RNA that has been particularly interesting is the family of small non-coding RNAs termed microRNAs (miRNAs). While miRNAs do not code for proteins, their biology is intimately intertwined, as miRNAs are estimated to regulate the majority of protein translation. This translational regulation stems from a miRNA’s ability to use sequence complementarity to identify a target messenger RNA and trigger translational suppression. Since miRNAs play crucial roles in biology, their dysregulation has been implicated in nearly every disease. As such, miRNAs are being evaluated as targets for therapeutic intervention. There are many different strategies being explored to manipulate these small RNAs. The furthest progressed strategy for miRNA manipulation is based on modified oligonucleotides and is being evaluated in the clinic; however, drugs developed using this approach have yet to receive FDA approval. While oligonucleotides are easy to design, obtaining optimal drug like properties has remained a challenge. Alternative strategies for miRNA manipulation, such as small molecules, have remained at the proof-of-concept stage largely due to the challenges associated with their discovery and design. To facilitate the discovery of new small molecules and strategies to manipulate miRNA biology, new technologies were developed. These reported techniques focused on the generation of RNA-protein conjugates that were crucial to the development of both high-throughput screening and biological assays. The first of these assays is focused on inhibiting the biogenesis of miRNAs and was able to identify natural product extracts as a source of small molecules capable of binding and disrupting miRNA precursors. The second assay platform discovered a small molecule capable of disrupting a known miRNA precursor-RNA binding protein interaction, pre-let-7d-Lin28. Having successfully disrupted a clinically relevant RNA-protein interaction, a final assay was created to aid in the discovery of new RNA-protein interactions by reporting direct interactions in live cells. Altogether, the technologies reported serve as a launching platform for the discovery of molecules with the ability to manipulate clinically significant miRNAs.