Ankenbruck, Nicholas
(2019)
Detection and Perturbation of MicroRNAs using Synthetic Chemical Probes.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
microRNAs (miRNAs) are small, non-coding RNA molecules capable of regulating protein expression in cells via binding to a complementary sequence within the 3’ untranslated region (3’ UTR) or coding domain sequence (CDS) of target messenger RNAs (mRNAs), thereby inducing translational repression and ultimately mRNA degradation. As such, its unsurprising that dysregulation of miRNAs has been implicated in a wide range of diseases in humans, including cancer. While regulation of miRNAs has largely been mediated by oligonucleotide reagents, current technologies exhibit limitations in terms of stability and pharmacological properties. In contrast, small molecules possess many advantageous qualities as tools to perturb miRNA function, including fast activity, systematic delivery, and enhanced cell permeability. Two luciferase-based reporters were developed into a cell-based assay employed in separate high-throughput screens of >300,000 compounds to identify selective small molecule modifiers of miR-21 or miR-122 function. The work presented herein discusses validation of oxadiazole, ether-amide, and N-acylhydrazone miR-21 inhibitors as well as sulfonamide and methanone inhibitors of miR-122 function identified in their respective high-throughput screens. In addition to secondary assays to confirm function and specificity of the lead molecules, structure-activity relationship (SAR) studies were carried out in order to determine important structural features of each scaffold and to attempt to improve their activity. Moreover, preliminary functional assays were carried out to evaluate therapeutic potential of improved molecules identified through the SAR study. Collaborations with start-up companies to discover small molecule inhibitors of miRNA processing are also presented. Additionally, delivery of DNA logic devices capable of recognizing miRNAs in cells or releasing a small molecule output in response to miRNA inputs are discussed. Optical regulation of chemical tools using light affords spatial and temporal control over biological processes and as such, a second-generation caged promoter was incorporated into a plasmid to afford optical control of transcription. Finally, methods for delivery of γ-modified peptide nucleic acids (PNAs) and morpholino oligomers into mammalian cells for use as splice-switching oligonucleotides or anti-miRNA agents were explored.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
| Title | Member | Email Address | Pitt Username | ORCID |
|---|
| Committee Chair | Deiters, Alex | | | | | Committee Member | Islam, Kabirul | | | | | Committee Member | Childers, Seth | | | | | Committee Member | Das, Subha | | | |
|
| Date: |
26 September 2019 |
| Date Type: |
Publication |
| Defense Date: |
26 April 2019 |
| Approval Date: |
26 September 2019 |
| Submission Date: |
13 June 2019 |
| Access Restriction: |
3 year -- Restrict access to University of Pittsburgh for a period of 3 years. |
| Number of Pages: |
524 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
microRNAs, small molecule modulator, DNA computation, optochemical tools, morpholino oligomers |
| Date Deposited: |
26 Sep 2019 20:49 |
| Last Modified: |
26 Sep 2022 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/36935 |
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