Date of Award

Spring 5-18-2024

Degree Type

Dissertation

Degree Name

PhD. Chemistry

Department

Chemistry and Biochemistry

Advisor

Gregory R. Wiedman, PhD

Committee Member

Gerald J. Buonopane, PhD

Committee Member

Cosimo Antonacci, PhD

Committee Member

Stephen Kelty, PhD

Keywords

Aptamers, Rifampicin, SELEX, Azobenzene, Tryptophan, Intrinsic

Abstract

Understanding fluorescence-based methods for detecting small biomolecules is a crucial area of focus in research investigations. Our studies focused on the challenge in two parts: Non-intrinsic fluorescence and intrinsic fluorescence. For studies on Non-intrinsic fluorescence, Rifampicin (Rif), an antibiotic harnessed for treating diverse bacterial infections, including tuberculosis (TB), serves as a focal point. Leveraging the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process, aptamers emerged from the nonspecific 40-mer Deoxyribonucleic Acid (DNA) library to interact with its target, Rif. During each SELEX cycle, the recovered nucleotides underwent amplification via Polymerase Chain Reaction (PCR) until the most selective aptamers were found. The core objectives of this study encompassed the discovery of aptamers targeting Rifampicin and the comprehension of their binding efficacy to Rif.

For intrinsic small-molecule fluorescence, photo-switchable molecules were explored to investigate the interaction between the azobenzene moiety and neighboring aromatic amino acids tryptophan. Under 365 nm irradiation, the photo-switching behavior favored a singular isomer, which is red-shifted or visibly fluorescent. This is the first evidence to suggest that primary protein structure could affect photo-switch activity. The insights from this investigation can potentially advance the understanding of photo-switch molecules within biomolecules.

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