Construction of deoxyribonucleic acid motors enabling single-molecule detection

癌症是加拿大男性和女性发病和死亡的主要原因。癌症的早期和准确检测不仅可以降低总体癌症死亡率和发病率，还可以减轻经济负担。开发基于生物标志物的癌症筛查工具是在早期阶段识别癌症的最有希望的方法之一。早期癌症诊断通常需要检测生物体液、癌细胞和组织中的超低水平生物标志物。大多数公认的癌症生物标志物是蛋白质。现有的技术都不能满足超灵敏检测蛋白质生物标志物和血液和组织中罕见异常细胞的技术要求。为了应对早期癌症检测的技术挑战，我建议开发新的单分子DNA运动系统，以实现(I)超痕量蛋白质的放大检测，（ii）癌细胞和组织上和内部特定生物标志物的成像，以及（iii）捕获和表征循环肿瘤细胞。我建议构建单分子DNA马达系统，它将通过蛋白质分子的特定结合而启动，并将产生数百到数千个定位在磁性纳米颗粒上的荧光分子。定位的荧光分子便于单分子检测。荧光显微镜将用于检测和成像单个纳米颗粒的荧光。由于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，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。所提出的系统将为检测低丰度蛋白质提供最终的灵敏度。我将开发两个互补的单分子DNA马达系统。第一个系统将利用切割酶的高切割活性在一到两个小时内实现快速的单分子检测，而第二个系统将利用DNA酶的切割活性进行单分子检测，从而避免了对蛋白质酶的需要。虽然第二个系统将比第一个系统慢，但它的操作将不使用蛋白质酶，并将在室温下进行，使其更适用于资源有限的情况。这项建议的研究将开发一个多功能工具箱，以解决早期癌症检测和癌症预后的分析挑战。该研究将拓宽DNA马达的分析应用，为单分子蛋白质检测开辟新的途径。除了检测蛋白质和癌细胞外，该系统还将适用于许多其他生物靶标，如核酸、脂质、低聚糖、病原体和病毒。该项目将为我的学员提供多学科的综合培训机会和一个刺激的学习和研究环境。

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.