Author ORCID Identifier

https://orcid.org/0000-0001-8041-1325

Semester

Fall

Date of Graduation

2025

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Aaron Robart

Committee Member

David Smith

Committee Member

Ivan Martinez

Committee Member

Michael Schaller

Committee Member

Bradley Webb

Abstract

The 10-23 DNAzyme is a catalytically active DNA oligonucleotide capable of cleaving RNA at purine-pyrimidine junctions in the presence of divalent metal ions, most notably magnesium. Despite its promise as a programmable RNA knockdown tool, its clinical and biotechnical utility has been hampered by limited structural characterization, low catalytic efficiency under physiological conditions, and poor understanding of its in-cell activity. This dissertation presents a comprehensive framework for the structural and functional analysis of DNAzymes using the 10-23 DNAzyme as a model system. In the absence of high-resolution structures, we first employed detailed in vitro biochemical assays to map essential residues within the catalytic core and assess turnover limitations imposed by substrate binding affinity and product inhibition. To address the lack of high-resolution structural information, X-ray crystallography and cryo-EM were employed, both with the design of protein scaffold systems to facilitate visualization of the DNAzyme catalytic core. In addition to these methods, dimethyl sulfate (DMS) labeling verified interactions and provided more dynamic and mechanistic information. Through these experiments, a novel mode of 10-23 DNAzyme activity was visualized operating through the interaction of an ‘activating loop’ within the catalytic core. Overall, this methodological framework provides a workflow that can not only be applied to other DNAzymes but toward any small structured nucleic acid motif, aiming to address the larger knowledge gap of nucleic acid structure that exists within the field.

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