Design, synthesis, and translational applications of fluorescent DNA repair probes

The process of DNA repair is critical to the viability of life on earth. However, our understanding of DNA repair is only as good as the tools we have to study it. While many of the traditional biochemical methods first pioneered in the 1960s and 1970s have yielded numerous important discoveries, they are often laborious and indirect. Efforts to study the biology and therapeutic relevance of DNA repair pathways can be limited by such method, and novel tools are required to keep up with the expanding capabilities of other fields. Significant advances in optics, microprocessors, electrical engineering, and nanofabrication have led to the widespread use of fluorescent microplate readers, fluorescence microscopes and flow cytometry instruments. However, such advances are meaningless without fluorescent assays that are compatible with such instruments. In addition to their compatibility with powerful new technologies, fluorescent probes offer the advantage of being able to directly assay for DNA repair activity in a simple, mix-andmeasure format. This work describes several efforts to design, synthesize and apply fluorescent probes of DNA repair. Chapter 1 summarizes the distinct classes of probe designs and their potential utility in varied research and preclinical settings. This work has been adapted in part with permission from: Wilson, D. L.; Kool, E. T. ACS Chem. Biol. 2018, 13 (7), 1721--1733. Copyright 2018 American Chemical Society. Chapter 2 presents work on a real-time probe of the DNA demethylase ALKBH2. The DNA repair enzyme ALKBH2 is implicated in both tumorigenesis as well as resistance to chemotherapy in certain cancers. It is currently under study as a potential diagnostic marker and has been proposed as a therapeutic target. To date, however, there exist no direct methods for measuring the repair activity of ALKBH2 in vitro or in biological samples. In this chapter, we report a highly specific, fluorogenic probe design based on an oligonucleotide scaffold that reports directly on ALKBH2 activity both in vitro and in cell lysates. Importantly, the probe enables the monitoring of cellular regulation of ALKBH2 activity in response to treatment with the chemotherapy drug temozolomide through a simple fluorescence assay, which has only previously been observed through indirect means such as qPCR and western blots. Furthermore, the probe provides a viable high‐ Index vi throughput assay for drug discovery. This work has been adapted in part with permission from: Wilson, D. L.; Beharry, A. A.; Srivastava, A.; O'Connor, T. R.; Kool, E. T. Angew. Chem. Int. Ed. 2018, 57 (39), 12896--12900. Copyright 2018 Wiley-VCH. Chapter 3 describes the design and synthesis of probes to be used a general DNA glycosylase sensing platform. DNA glycosylases constitute a biologically and biomedically important group of DNA repair enzymes responsible for initiating base excision repair (BER). Measuring their activities can be useful for studying the mechanisms DNA damage and repair and for practical applications in cancer diagnosis and drug screening. Previous fluorescence methods for assaying DNA glycosylases are often complex and/or limited in scope to a single enzyme type. In this chapter we describe a universal base excision reporter (UBER) fluorescence probe design that implements an unprecedentedly rapid oxime reaction (> 150 M--1 s --1 ) with high specificity for the abasic (AP) site of DNA. The molecular rotor design achieves a robust > 250--500-fold increase in fluorescence upon reaction with AP sites in DNA. By using the fluorescence reporter in concert with specific DNA lesion-containing substrates, the UBER probe can be used in a coupled assay in principle with any DNA glycosylase. We demonstrate the utility of the UBER probe by assaying five different glycosylases in real time as well as profiling glycosylase activity in cell lysates. This work has been adapted in part with permission from: Wilson, D. L.; Kool, E. T. J. Am. Chem. Soc. 2019, 141 (49), 19379--19388. Copyright 2019 American Chemical Society. Chapter 4 explores preliminary data on a broad range of downstream applications for the UBER probe system outside of sensing DNA glycosylase activity including activator/inhibitor screening, AP site sensing in live cells, and nucleic acid sensing. Additionally we propose several other applications to exploit the unique chemical and photophysical properties of the UBER probes described in chapter 3

Keywords: Chemistry.
Advisor: Kool Eric T.
Rao Jianghong
Xia Yan