Molecular Engineering of Deoxyribozymes for Amplified Protein Detection and Cell Imaging

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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.**