Molecular Engineering of Deoxyribozymes for Amplified Protein Detection and Cell Imaging

蛋白质是所有生物的重要组成部分，执行从表达基因组信息到维持动态平衡的广泛功能。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法、Western印迹法、免疫-PCR法)通常需要复杂的仪器、繁琐的操作和训练有素的具有专门技术的人员，因此不适合许多实际应用，如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应对蛋白质分析中的当前挑战，应用范围从基础研究到个性化医疗保健。 在这项建议中，我们的目标是通过脱氧核酶(DNAzymes)的分子工程来开发新的信号转导和放大机制，用于敏感和简便的蛋白质分析。具体地说，我们已经确定了我们打算重点研究的两个核心领域。在一个领域，利用结合诱导的DNA链置换技术，DNAzyme将被设计成蛋白质响应传感器。一组这样的传感器将被创建并定制成均质荧光分析，用于分析人类血清样本中的多种癌症生物标记物。还将通过将蛋白质响应型DNAzyme传感器与DNA功能化的金纳米颗粒相结合，开发适用于疾病诊断的均质比色分析方法。在第二个领域，DNAzyme将被设计成实时传感器，通过结合诱导DNA杂交来成像细胞表面的蛋白质表达。无需任何清洗步骤即可产生用于细胞成像的敏感荧光信号的能力，将使这套新型传感器有别于任何现有的免疫染色试剂。这一特殊功能还将允许实时监测活细胞细胞膜上的蛋白质变化。将开发一组这样的传感器，用于监测乳腺癌标记物，特别是乳腺癌干细胞标记物的表达。 在我们提出的研究计划中，新的信号放大机制具有高度的通用性，因此将能够检测到几乎任何具有两个或更多亲和配体的目标蛋白。因此，可以很容易地定制它们来检测人类、生物或环境样本中的生物标记物。在不需要任何分离、热循环或酶放大的情况下，能够产生灵敏的荧光或比色信号，这使得它比许多传统的在护理环境或细胞水平上分析蛋白质的方法更具优势。该项目的多学科性质也将为高素质人才的培训提供一个丰富的环境。

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.