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Development of small molecule modifiers of microRNA functions and targeted activation of caged morpholino oligomers

Kumbhare, Rohan (2021) Development of small molecule modifiers of microRNA functions and targeted activation of caged morpholino oligomers. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

MicroRNAs (miRNAs) are small non-coding RNA molecules (~22-nucleotides long) and constitute up to 60% of human genes. miRNAs are primarily involved in gene silencing by binding to the 3’-untranslated region (3’-UTR) of messenger RNAs (mRNAs), leading to translational inhibition or mRNA cleavage. The liver-specific miR-122 plays a critical role in the replication of Hepatitis C virus (HCV) while the progression of many cancer types is affected by the downregulation of miR-21. The first part of the thesis focuses on the design, synthesis, and identification of small molecule inhibitors of miR-122 and miR-21. Upon identification of the hit molecules identified by high throughput screening of small molecule libraries by, previously developed, miR-122 and miR-21 reporter assays, structure-activity relationship (SAR) studies were undertaken and lead molecules exhibiting improved activity were selected. The lead molecules were further explored for their mode of action in the modulation of miR-122 and miR-21.
In the second part of the thesis, strategies for selective activation of gene regulation by morpholino oligomers (MOs) were explored. Topologically constrained or caged MOs exhibit poor to none target sequence binding by Watson-Crick base-pair complementarity. Caging of MOs can be achieved by construction of bi-functional linkers for joining both the ends of linear MOs. Gene silencing with spatiotemporal resolution can be achieved by targeted activation and linearization of these caged MOs. In this study, a number of projects with small molecule-mediated and selective enzyme (β-lactamase)-mediated activation strategies were undertaken.
In the final part of the thesis, other methodologies utilizing light activation to mediate gene regulation were explored. The maturation process of nascent mRNAs proceeds by attaching the mRNA 5’-end with a modified guanosine triphosphate cap and polyadenylation on the 3’-end. The mRNA translation can be modulated by modifications to the 5’-cap as the cap interactions with initiation factors like eIF4E are crucial for the assembly of translational machinery. A closer look at eIF4E and 5’-cap interactions revealed multiple positions of caging with a photocleavable group for light-mediated activation of mRNA translation. Strategies involving the incorporation of two caged cap analogs and caged guanosine triphosphate were pursued.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Kumbhare, Rohanrok42@pitt.edurok42
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee MemberFloreancig, Paulflorean@pitt.edu
Committee MemberHorne, Sethhorne@pitt.edu
Committee MemberDas, Subhasrdas@andrew.cmu.edu
Committee ChairDeiters, Alexanderalexdeiters@gmail.com
Date: 20 January 2021
Date Type: Publication
Defense Date: 18 June 2020
Approval Date: 20 January 2021
Submission Date: 6 September 2020
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 318
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: miR-122, miR-21, SAR, small molecule, medicinal chemistry, HCV, cancer, ntla, morpholino oligomers, tetrazine, trans-cyclooctenol, mRNA 5'-cap, photocaging group, cap analog
Date Deposited: 20 Jan 2021 18:53
Last Modified: 20 Jan 2021 18:53
URI: http://d-scholarship.pitt.edu/id/eprint/39714

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