Construction of deoxyribonucleic acid motors enabling single-molecule detection

构建可实现单分子检测的脱氧核糖核酸马达

• 批准号: RGPIN-2019-06363
• 负责人: Zhang, Hongquan
• 金额: $ 2.62万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=757980
• 关键词: Construction; deoxyribonucleic; acid; motors; enabling

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.

癌症是加拿大男女发病和死亡的主要原因。癌症的早期和准确检测不仅有望降低总体癌症死亡率和发病率，而且还有望降低经济负担。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前途的方法之一。早期癌症诊断通常需要检测生物体液、癌细胞和组织中超低水平的生物标志物。大多数公认的癌症生物标志物是蛋白质。现有技术均不符合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。 为了应对早期癌症检测的技术挑战，我建议开发新的单分子DNA马达系统，这将使（i）超痕量蛋白质的放大检测，（ii）癌细胞和组织上和癌细胞和组织中的特定生物标志物的成像，以及（iii）循环肿瘤细胞的捕获和表征。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异性结合而启动，并产生位于磁性纳米颗粒上的数百至数千个荧光分子。局部荧光分子促进单分子检测。荧光显微镜将用于检测和成像单个纳米颗粒的荧光。因为单个蛋白质分子的结合引发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。第一个系统将利用切口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，从而避免了对蛋白酶的需要。虽然第二个系统将比第一个慢，但它的操作将不使用蛋白酶，并将在室温下进行，使其更适用于资源有限的情况。 这项拟议的研究将开发一个多功能工具箱，用于应对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，并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之外，所提出的系统还将适用于许多其他生物目标，例如核酸、脂质、寡糖、病原体和病毒。拟议的研究将为我的学员提供综合的多学科培训机会和激励性的学习和研究环境。