Electrochemical Measurements on Self-Assembled Monolayers of DNA to Follow Anti-Cancer Drug Activity and Helicase Interactions

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2018-05

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Abstract

Biological study would benefit greatly from techniques that detect cellular processes typically constrained within cells. Electrochemistry using DNA-mediated charge transport enables studies of DNA interactions with proteins and drugs. We developed DNA-based multiplexed chips to mimic cellular environments or operate in cellular extracts. In this dissertation, electrochemical measurements were performed with chips bearing monolayers of DNA to follow anticancer drug activity and DNA-helicase interactions. Many cancer treatments involve DNA damage, and understanding these drugs involves controlling activation pathways and precisely following DNA damage repair. We designed an electrochemical chip of DNA modified electrodes to follow DNA damage, offering benefits over gel electrophoresis assays. We used chips to study the anticancer agent β-lapachone (ß-lap), which generates DNA damaging peroxide in the presence of overexpressed NAD(P)H: quinone oxidoreductase 1 (NQO1), a hallmark of many cancer cells. Initially, ß-lap was studied in a model system reproducing certain pathways of drug activation, DNA damage repair, and drug abrogation. We observed drug-specific changes from these chips and demonstrated a high correlation with the ß-lap-induced redox cycle.Our study revealed significant signal changes at clinically relevant levels and sub-lethal concentrations. Catalase, an enzyme decomposing peroxide, suppressed signal changes under conditions specific to cancer. Thus, this chip-based platform enabled unique tracking of ß-lap-induced DNA damage repair. Subsequently, we followed ß-lap activity in cellular lysates with these devices to correlate cell death activity with DNA damage. Cells were prepared to be proficient or deficient in NQO1 to mimic cancerous and healthy cells. Cells were lysed and added to chips, and β-lap activity was followed by signal changes arising from DNA damage. Devices showed an approximate fourfold difference in electrochemical response to NQO1+ over NQO1− cells, as well as great selectivity to controls deactivating the drug-induced DNA damage pathways. Saturation of DNA damage on the chip correlated with the onset of cell death from viability assays. These devices could be applied for screening of multiple anticancer drugs from small samples to guide cancer treatment.

Xeroderma pigmentosum group B (XPB) is an essential helicase involved in both DNA repair and transcription. Significant characteristics of XPB binding and activity remains to be established. We utilized DNA electrochemistry to sense the DNA-helicase interactions of three distinct XPB helicases. Changes in DNA duplex stability were quantified upon helicase binding. Binding dissociation constants were estimated in the range from 10-50 nM. and ATP-stimulated DNA unwinding activity was followed, revealing distinct modes of operation confirmed by crystal structures. These devices provided a sensitive measure of the structural thermodynamics and kinetics of DNA-helicase interactions. Chapter 1 introduces DNA, electrochemistry, and the specific field of DNA electrochemistry. Chapter 2 relates our research of β-lap in a model system incorporating the drug activation cycle, DNA base-excision repair by a glycosylase, and a drug abrogation pathway. Chapter 3 builds on this study to investigate β-lap activity in cellular lysates with differing concentrations of NQO1 that were proficient or deficient in DNA damaging activity. Chapter 4 describes the DNA binding and unwinding activity of XPB helicases with DNA devices.

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Electrochemical sensors, DNA repair, Biosensors, DNA helicases

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