Date of Award

May 2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Xiaohua Peng

Committee Members

Andy Pacheco, Alan Schwabacher, Alexander Arnold, Mark Dietz

Keywords

Coumarin, DNA Interstrand Cross-links, Ligation Reactions, Mutation Detection, Site-Specific Labeling

Abstract

Coumarin derivatives have been widely utilized as cross-linking agents in polymer science, being fluoroprobes in biochemistry and as medicines in pharmacy. But the coumarin's fluorogenic properties and reactivities in DNA were rarely reported and unclear, which limits its bioapplications due to possible side reactions towards biomolecules. In this thesis, we investigated the activity of coumarin moiety towards natural DNA and expanded its application in DNA-associated study. We have found that coumarin derivatives can serve as perfect DNA cross-linking agents, as alkylation agents for site-specific labeling, and fluoroprobes for single nucleotide polymorphism (SNP) analysis, which provided a novel insight of biotoxicity of coumarin in biological system and a novel bioanalytical tool.

First, sequence-dependent DNA-templated fluorogenic "click" reaction has been developed for SNP detection. Oligonucleotide (ODN) probes containing nonfluorescent alkyne-modified coumarin or azide group at the terminal sites efficiently hydride with the matched ODN template, a tumor-associated p53 gene with single nucleotide transition from dT to dC. The alkyne and azide groups at the adjacent position provided effective concentration for fluorogenic "click" reaction which generated substantial fluorescence signal. However, no obvious fluorescence was observed for noncomplementary DNA templates. In this way, coumarin-based fluorogenic "click" reaction can be used for SNP analysis. In addition, the coumarin derivatives with the bromo group were employed as alkylation agents for site-specifically labeling dT in natural DNA, affording highly fluorescent ODNs for bioanalysis.

Second, we have found that the coumarin moieties induced quantitative photoreversible DNA interstrand cross-link (ICL) formation with dT allowing for real-time monitoring DNA cross-linking process via fluorescence assay. Photoinduced [2+2] cycloaddition between coumarin moiety and thymidine upon 350 nm irradiation generated DNA ICLs with formation of syn- and anti-cyclobutane adducts, which can be reversed into the single stranded ODNs with 254 nm photoirradiation. ICL formation completely quenched the fluorescence of coumarin, which, for the first time, enables fast and real-time detection of DNA cross-linking and photoreversibile process via fluorescence spectroscopy.

Finally, rearrangement of the kinetic-controlled ligation products to the thermo-controlled ICL products was observed during photoswitchable process. In addition, light-sensitive and sequence-specific photorelease of coumarin moieties from ODN strand was discovered. Further study showed that the G:C base pairs and 350 nm irradiation played a central role in the reaction.

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