Abstract

G-quadruplexes are noncanonical secondary structures that form in guanine-rich nucleic acid sequences and are thought to be involved in the control of gene expression.1 Unfortunately, it has been difficult to prove that these structures exist in vivo because of their dynamic nature. Rather than try to develop G-quadruplex specific binders as others have done,2 our idea is to photochemically trap certain types of G-quadruplexes by irreversible photoproduct formation3–5 and then detect the stable G-quadruplex-specific photoproducts by Isotopic Dilution Mass Spectrometry (IDMS).6 This IDMS method could then be used to unambiguously confirm the presence of specific types of G-quadruplexes in vivo and help justify more detailed studies of their role in the regulation of DNA transcription and gene regulation.7,8 Our approach is based on the observation that UV-irradiation produces DNA photoproducts that form with stereochemistry and regiochemistry that depend on the structure of the DNA and the relative positioning and orientation of the precursor bases.9 While the primary photoproduct formed in duplex DNA is an adjacent cis,syn cyclobutane pyrimidine dimer (CPD) with a head-to-head orientation,10 non-adjacent anti CPDs will form in basket and chair-type G-quadruplexes with a head-to-tail orientation.5 These adjacent and non-adjacent CPDs can be detected by enzymatic degradation with nuclease P1 and snake venom phosphodiesterase, which produces a dinucleotide CPD with an intradimer phosphodiester linkage from adjacent CPDs, and dinucleotide CPDs lacking a phosphodiester from non-adjacent CPDs.9 These enzymatically degraded DNA photoproducts can be readily distinguished by HPLC and mass spectrometry, making them suitable as intrinsic photoprobes of B and non-B DNA conformations. Herein, we report methods for preparing isotopically labeled cyclobutane thymidine dimers and show how these standards can be used to identify and quantify non-adjacent photoproducts produced in non-B DNA structures such as G-quadruplexes by IDMS. We therefore expect that this method should find application to the study of the photochemistry and photobiology of non-B DNA structures in vivo.

Committee Chair

John Taylor

Committee Members

Michael Gross; Hani Zaher; Jennifer Heemstra; 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

8-6-2025

Language

English (en)

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Chemistry Commons

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