Construction of deoxyribonucleic acid motors enabling single-molecule detection

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Cancer is the leading cause of morbidity and mortality in Canadian men and women. Early and accurate detection of cancer promises to reduce not only overall cancer mortality and morbidity but also the economic burden. Developing biomarker-based tools for cancer screening represents one of the most promising approaches to identifying cancer in its early stages. Early cancer diagnosis often entails detecting ultra-low levels of biomarkers in biological fluids, cancer cells, and tissues. The majority of the recognized cancer biomarkers are proteins. None of the available technologies meet the technical requirements for ultra-sensitive detection of protein biomarkers and rare abnormal cells in blood and tissues. To confront the technical challenges of early cancer detection, I propose to develop novel single-molecule DNA motor systems that will enable (i) amplified detection of ultra-trace amounts of proteins, (ii) imaging of specific biomarkers on and in cancer cells and tissues, and (iii) capture and characterization of circulating tumor cells. I propose to construct single-molecule DNA motor systems that will be turned on by the specific binding of a protein molecule and will produce hundreds to thousands of fluorescent molecules localized on a magnetic nanoparticle. The localized fluorescent molecules facilitate single-molecule detection. A fluorescence microscope will be used to detect and image the fluorescence of individual nanoparticles. Because the binding of a single protein molecule initiates the generation of the fluorescent molecules on the nanoparticle, imaging of the individual nanoparticles represents single-molecule detection of the initial binding protein. The proposed systems will provide the ultimate sensitivity for detection of low-abundance proteins. I will develop two complementary single-molecule DNA motor systems. The first system will utilize the high cleaving activity of nicking enzymes to achieve fast single-molecule detection within one to two hours, whereas the second system will harness the cleaving activity of DNA enzymes for single-molecule detection, obviating the need for protein enzymes. Although the second system will be slower than the first one, its operation will not use protein enzymes and will be carried out at room temperature, making it more practical for resource-limited scenarios. This proposed research will develop a versatile toolbox for tackling analytical challenges in early cancer detection and cancer prognosis. The research will broaden the analytical applications of DNA motors and pave a new way to single-molecule protein detection. Beyond detection of proteins and cancer cells, the proposed systems will also be applicable to many other biological targets, such as nucleic acids, lipids, oligosaccharides, pathogens, and viruses. The proposed research will offer integrated multidisciplinary training opportunities and a stimulating learning and research environment for my trainees.