Construction of deoxyribonucleic acid motors enabling single-molecule detection

癌症是加拿大男性和女性发病率和死亡率的主要原因。及早、准确地发现癌症，不仅可以降低癌症的总体死亡率和发病率，还可以减少经济负担。开发基于生物标记物的癌症筛查工具是在癌症早期阶段识别癌症最有希望的方法之一。癌症的早期诊断通常需要检测生物液、癌细胞和组织中超低水平的生物标记物。大多数公认的癌症生物标记物是蛋白质。现有的技术都不能满足超灵敏检测血液和组织中蛋白质生物标志物和罕见异常细胞的技术要求。为了应对癌症早期检测的技术挑战，我建议开发新型的单分子DNA马达系统，该系统将能够(I)放大检测超微量蛋白质，(Ii)对癌细胞和组织及其内部的特定生物标记物进行成像，以及(Iii)捕获和表征循环中的肿瘤细胞。我建议构建单分子DNA马达系统，该系统将通过蛋白质分子的特定结合而启动，并将产生成百上千个定位在磁性纳米颗粒上的荧光分子。局域化的荧光分子便于单分子检测。将使用荧光显微镜来检测和成像单个纳米颗粒的荧光。因为单个蛋白质分子的结合启动了纳米颗粒上荧光分子的产生，所以单个纳米颗粒的成像代表了对初始结合蛋白的单分子检测。所提出的系统将为低丰度蛋白质的检测提供最终的灵敏度。我将开发两个互补的单分子DNA马达系统。第一个系统将利用划痕酶的高切割活性在一到两个小时内实现快速单分子检测，而第二个系统将利用DNA酶的切割活性进行单分子检测，从而消除对蛋白质酶的需求。虽然第二个系统的运行速度会比第一个系统慢，但它的运行不会使用蛋白质酶，而且将在室温下进行，使其更适用于资源有限的场景。这项拟议的研究将开发一个通用的工具箱，以应对癌症早期检测和癌症预后方面的分析挑战。该研究将拓宽DNA马达的分析应用，为单分子蛋白质的检测开辟新的途径。除了对蛋白质和癌细胞的检测外，所提出的系统还将适用于许多其他生物靶标，如核酸、脂类、寡糖、病原体和病毒。拟议的研究将为我的受训人员提供综合的多学科培训机会和激励的学习和研究环境。

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.