A versatile protein platform for sequence specific nucleic acid detection

Abstract

Detection and quantification of genetic material is fundamental for many research and diagnostic applications. Many different methods has been developed towards this objective, where each is typically tailored to detect a specific types of nucleic acid target under defined conditions. We have devised a platform technology based on a single-protein probe architecture compatible with detection of all types of genetic materials (RNA, ssDNA, dsDNA), to achieve design flexibility and compatibility with existing biological assays, ease of mass production and quality control for clinical applications, and establish direct link between sequence recognition and signal generation.In this dissertation, I describe the development of a universal platform for sequence-specific nucleic acid imaging and detection based on Transcription Activator-Like Effector (TALE) proteins with versatile functional domains, characterization of the platform, and its application in live cells, fixed cellular specimens, and cell-free context. In fixed and live cell specimens, I used TALE probes to robustly label individual genome loci under most native nucleic acid condition in the cell environment. The engagement of TALEs with genomic DNA is minimally disruptive to chromatin and is analogous to the interaction of endogenous transcription factors with their target sites. Furthermore, because TALEs are pure, single-component protein probes, they can be easily engineered to incorporate any protein modality used for visualization. These properties render TALEs an attractive and minimally invasive platform for revealing native genomic structure and function by imaging. TALE architecture can also be used for ex vivo nucleic acid detection in cell-free context. Nucleic acid detection methods have facilitated the development of biological basic research and served as a core technique in diagnostics for pathogens in various biological samples. I deployed TALE probes with various reporter units to implement sequence-specific nucleic acid detection in biological samples. The detection is highly sensitive and comparable detection limit with contemporary assays with robust expansion to multiplexing targeting. Thus, the TALE platform provides a useful and versatile new way to understand the spatial and temporal aspects of nucleic acid in cell environment, and a corresponding new platform and technology for nucleic acid detection in clinical or other biological samples.