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Conditional Control of Oligonucleotides in Gene Expression and DNA Computation, and Targeted Protein Inactivation

Bardhan, Anirban (2024) Conditional Control of Oligonucleotides in Gene Expression and DNA Computation, and Targeted Protein Inactivation. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Conditional control of biomolecules is critical for introducing perturbations into dynamic biological processes to investigate them. Synthetic oligonucleotides are versatile tools, when conditionally controlled, allows for regulation of gene expression, gene editing and DNA computation. Light and small molecules are external triggers that allow activation of such oligonucleotides offer spatial and temporal control over such processes. Herein, I describe my contribution toward: (1) Development of new red-shifted photoactivatable caging groups and extensive study of their photophysical properties, leading to repurposing of common UV-activatable caging groups for efficient violet light activation, (2) development of nucleobase caged photoactivatable morpholino oligonucleotides for use as knockdown reagents in sea urchin embryos, (3) development of nucleobase caged small molecule activatable morpholino oligonucleotides for regulating gene expression in mammalian cells and aquatic embryos (4) development of a photoactivatable cyclic morpholino oligonucleotide for optically controlling function of a critical microRNA in zebrafish embryos (5) development of second generation small molecule activation through DNA computation, though use of multistrand reporter gate and a novel hairpin reporter gate and (5) development of a targeted light inactivation approach for recombinant proteins through localized oxidation via site-specific installation of photosensitizers.
Additionally, I have contributed to the development of photocaged guide RNA (gRNA) for demonstration of optical control of gene editing in mammalian cells and zebrafish embryos using the CRISPR/Cas9 system. Moreover, the caged gRNAs have been applied to control activation of transcription and base editing. Furthermore, I have contributed to the development of an optically controlled Boolean operation in the form of NOT gate, which ensures precise and accurate computation of the NOT logic function through use of light as trigger.

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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Bardhan, Anirbananb229@pitt.eduanb229
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairDeiters, AlexanderDEITERS@pitt.edu
Committee MemberFloreancig, Paulflorean@pitt.edu
Committee MemberHorne, Sethhorne@pitt.edu
Committee ChairArmitage, Brucearmy@andrew.cmu.edu
Date: 8 January 2024
Date Type: Publication
Defense Date: 9 August 2022
Approval Date: 8 January 2024
Submission Date: 29 August 2023
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 355
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: Organic Chemistry, Chemical Biology
Date Deposited: 08 Jan 2024 18:07
Last Modified: 08 Jan 2024 18:07
URI: http://d-scholarship.pitt.edu/id/eprint/45406

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