Molecular Engineering of Deoxyribozymes for Amplified Protein Detection and Cell Imaging

蛋白质是所有生物体的基本组成部分，执行从表达基因组信息到维持体内平衡的大量功能。它们也是一类重要的疾病诊断生物标志物。目前的蛋白质检测技术（例如，ELISA、蛋白质印迹、免疫-PCR）通常需要复杂的仪器、繁琐的操作和具有专门技术的训练有素的人员，因此不适合许多实际用途，例如护理点疾病诊断和高通量分析。我们的研究计划将专注于通过开发新的化学和生物工具来解决蛋白质分析中的当前挑战，其应用范围从基础研究到个性化医疗保健。在这个建议中，我们的目标是开发新的信号转导和放大机制，通过分子工程的脱氧核酶（DNAzymes）的敏感和简便的蛋白质分析。具体来说，我们已经确定了两个核心研究领域，我们打算集中在一个领域，DNA酶将被改造成蛋白质响应传感器使用结合诱导的DNA链置换技术。一组这样的传感器将被创建并定制成同质荧光检测，用于分析来自人类血清样本的多种癌症生物标志物。还将通过将蛋白质响应性DNAzyme传感器与DNA功能化金纳米颗粒相结合来开发适用于护理点疾病诊断的均相比色测定。在第二个领域，DNA酶将被设计成实时传感器，通过结合诱导的DNA杂交对细胞表面的蛋白质表达进行成像。在没有任何洗涤步骤的情况下产生用于细胞成像的灵敏荧光信号的能力将使这组新的传感器与任何现有的免疫染色试剂区分开来。这一特殊功能还将允许实时监测活细胞的细胞膜上的蛋白质变化。将开发一组这样的传感器，用于监测乳腺癌细胞系中癌症标志物，特别是癌症干细胞标志物的表达。在我们提出的研究计划中，新的信号放大机制是高度通用的，因此将能够检测几乎任何具有两个或更多个亲和配体的靶蛋白。因此，它们可以很容易地定制，以检测来自人类，生物或环境样品的生物标志物。在不需要分离、热循环或酶扩增的情况下产生灵敏的荧光或比色信号的能力使其优于许多传统方法，用于在护理点设置或在细胞水平分析蛋白质。该计划的多学科性质也将为高素质人才的培训提供丰富的环境。**

Proteins are essential components of all living organisms, performing a vast array of functions ranging from expressing the genomic information to maintaining homeostasis. They are also a class of important biomarkers for disease diagnostics. Current protein detection techniques (e.g., ELISA, Western blot, immuno-PCR) typically require complicated instrumentation, tedious operations, and well-trained personnel with specialized techniques, and thus are not amenable for many practical uses, such as point-of-care disease diagnostics and high throughput analysis. Our research program will focus on addressing current challenges in protein analysis by developing new chemical and biological tools with applications ranging from fundamental research to personalized healthcare.***In this proposal, we aim to develop novel signal transduction and amplification mechanisms through molecular engineering of deoxyribozymes (DNAzymes) for sensitive and facile protein analysis. Specifically, we have identified two core area of research we intend to focus on. In one area, DNAzymes will be engineered into protein-responsive sensors using binding-induced DNA strand displacement techniques. A panel of such sensors will be created and tailored into homogeneous fluorescent assays for analyzing multiple cancer biomarkers from human serum samples. Homogeneous colorimetric assays that are amenable for point-of-care disease diagnostics will also be developed by combining protein-responsive DNAzyme sensors with DNA functionalized gold nanoparticles. In the second area, DNAzymes will be engineered into real-time sensors for imaging protein expressions from cell surfaces by using binding-induced DNA hybridization. The ability to generate sensitive fluorescent signal for cell imaging without any washing steps will distinguish this novel set of sensors from any existing immunostaining reagents. This special feature will also allow real-time monitoring of protein changes on cell membranes from live cells. A panel of such sensors will be developed for monitoring expression of cancer markers, particularly cancer stem cell markers, from breast cancer cell lines.***The novel signal amplification mechanisms in our proposed research program are highly versatile, and thus will enable detection of almost any target protein with two or more affinity ligands. Therefore, they can be easily tailored to detect biomarkers from human, biological, or environmental samples. The ability to generate sensitive fluorescence or colorimetric signal without any requirement for separation, thermal cycling, or enzyme amplification, makes it advantageous over many traditional methods for analyzing proteins at point-of-care settings or at cellular levels. The multidisciplinary nature of this program will also provide an enriching environment for the training of highly qualified personnel.**