Date of Award

8-2025

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Kelly Teske, Ph.D.

Second Advisor

Frederick Stull, Ph.D.

Third Advisor

Alex Davies, Ph.D.

Fourth Advisor

John Spitsbergen, Ph.D.

Keywords

Assay, fluorescence polarization, high throughput, microRNA, RNA binding proteins

Abstract

Therapeutics targeting RNA is a rapidly growing field. While only 1.5% of the human genome encodes genes for protein, over 70% o=f the human genome contains the genes for noncoding RNAs that regulate protein expression and function. Expanding the paradigm of small molecule drug discovery from traditional protein targets to include RNA would broaden the therapeutic landscape and provide new methods to modulate previously undruggable targets. miRNAs (miRs) have been found to be aberrantly expressed in many disease states, including neurodegenerative diseases and cancers making them promising therapeutic targets. The production of functional, mature miRs requires multiple proteins in a biogenesis pathway and there are many auxiliary RNA-binding proteins (RBPs) that facilitate this biogenesis. These miR-RBP interactions represent potential small molecule targets to halt miR biogenesis, however limited high throughput screening (HTS) assays have been developed for this purpose. This work presents the adaptation of a fluorescence polarization (FP) assay, commonly used in protein-protein interactions, to the pri-miR-18a—hnRNP A1 interaction. The development, optimization, and initial HTS of 9,280 compounds was performed using this FP assay. Additionally, the development of a chemiluminescence-based assay, the catalytic-enzyme linked click chemistry assay (cat- ELCCA) was explored for orthogonal screening of small molecule modulators of the pri-miR18a— hnRNP A1interaction. The pri-miR-18a—hnRNP A1 interaction has no reported HTS assays developed to date so the optimization of the FP assay for this interaction can now be utilized in future HTS campaigns to identify small molecule modulators of the pri-miR-18a—hnRNP A1 interaction.

Access Setting

Dissertation-Open Access

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Chemistry Commons

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