Abstract

The structural complexity of nucleic acids plays a crucial role in genomic stability, gene regulation, and RNA functionality, yet studying their higher-order structures (HOS) and chemical modifications remains challenging due to their dynamic nature. This thesis employs mass spectrometry-based approaches—particularly ion mobility mass spectrometry (IM-MS) and MS-based footprinting—to probe DNA and RNA structures at the molecular level, with a focus on two systems: DNA photoadducts and messenger RNA (mRNA). Additionally, the study extends to protein systems, emphasizing the role of higher-order protein structure (HOS) in biological functions. In Chapter 2, we investigate the formation of cyclobutane pyrimidine dimers (CPDs) induced by UV irradiation in DNA, which have significant implications for skin cancer. Using IM-MS and MS/MS, we differentiate six stereoisomers of thymidine dimers (cis/syn vs. trans/anti), offering insights into their structural and ion mobility characteristics. The high resolution of IM-MS, complemented by fragmentation data from MS/MS, enables the baseline separation and precise characterization of these DNA photoproducts, advancing the understanding of UV-induced DNA damage. Building on these findings, Chapter 3 explores the application of trapped ion mobility spectrometry time-of-flight mass spectrometry (TIMS-TOF) to resolve closely related isomeric thymidine dimers with greater sensitivity and resolution. We utilize TIMS-TOF to completely resolve all six distinct isomers of thymidine dimers, enhancing our ability to characterize DNA conformational dynamics, particularly in the context of G-quadruplex (G4) structures. This data is compared and validated with existing isotopic dilution mass spectrometry (IDMS) results from Dr. Taylor's lab, providing further corroboration of our findings. In Chapter 4, we transition to the study of RNA higher-order structures, specifically focusing on mRNA. Traditional biophysical techniques provide valuable global structural insights but lack site-specific resolution. In contrast, our new mass spectrometry-based chemical footprinting strategy combines backbone- and base-specific reagents to probe RNA structure. This approach, demonstrated on the PreQ1 riboswitch, reveals structural changes upon ligand binding, providing a powerful and amplification-free method for probing RNA-ligand interactions. The method holds potential for advancing RNA-based research, with applications in RNA therapeutics, including mRNA vaccines. Finally, Chapter 5 shifts focus to the structural characterization of proteins, specifically Apolipoprotein E4 (ApoE4), a protein variant associated with Alzheimer’s disease. Using fast photochemical oxidation of proteins (FPOP) and mass spectrometry-based footprinting, we examine the conformational differences between ApoE4 and its engineered variants. Our findings reveal how mutations affecting oligomerization interfaces influence solvent accessibility and lipid-binding properties, offering new insights into the structural basis of ApoE4’s role in disease. By integrating advanced mass spectrometry techniques for the structural analysis of DNA, RNA, and proteins, this dissertation provides a comprehensive framework for investigating higher-order structures across these biomolecular systems. The methodologies developed here offer transformative opportunities for understanding nucleic acid and protein dynamics and have broad implications for biomedical research, including drug design, disease understanding, and therapeutic development.

Committee Chair

Michael Gross

Committee Members

Chun-Kan Chen; John-Stephen Taylor; Michael Gross; Timothy Wencewicz

Degree

Doctor of Philosophy (PhD)

Author's Department

Chemistry

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-14-2025

Language

English (en)

Author's ORCID

https://orcid.org/0009-0000-4920-9380

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Available for download on Friday, August 13, 2027

Included in

Chemistry Commons

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