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Development of a molecular sensing platform technology based on proximity proteolysis reaction
- Author(s)
- 박현지
- Advisor
- 유태현
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2021-02
- Language
- eng
- Keyword
- Biosensor; Protease; Protein engineering; Protein-DNA conjugate; Zymogen
- Alternative Abstract
- Nucleic acids, proteins, and small molecules either produced by or introduced into organisms are significant indicators for the evaluation of biological systems. Various methods have been developed to quantify these molecules. However, these methods were designed based on the properties of each type of molecule, inevitably resulting in the development of diverse technologies. Besides, the methods usually require complicated procedures, sophisticated instruments, and experts to detect and analyze the signals. Owing to the lack of generality and the complexity of the detection process, conventional methods may be limited for developing rapid and simple on-site assays. Here, I present a proximity proteolysis reaction (PPR) as a simple and universal detection platform. This reaction involves binders that recognize molecules of interest and enzymes—zymogen and protease—that produce measurable signals. As enzymes are linked to binders, recognition of binders by the analyte induces proteolysis of the zymogen by the protease, via colocalization of the enzymes at the analyte. The zymogen is activated because of the proximity effect, and the active enzyme generates a detectable signal via hydrolysis of its substrate. The reaction was applied to develop PPR-based homogeneous assay methods for the detection of DNAs, RNAs, proteins, antibodies, and small molecules. Single-stranded DNAs (ssDNAs) were utilized as binders for target recognition and as linkers that allow the tethering of enzymes and binders. Through protein engineering and chemical conjugation methods, a process was established to produce the desired bioconjugates as detecting molecules. To improve the generality of the method for detecting nucleic acids, a signal converter was devised. Additionally, a signal amplification process was designed to achieve a high sensitivity of the assay. ssDNAs, antibodies, aptamers, and antigens including proteins and small molecules showed acceptability as a binder, and replacement of the binder led to the development of detection methods for a wide range of molecules. The assays were conducted in a single step at a constant temperature, and colorimetric signals, which can be measured by simple instruments, were detected in one hour. Despite the simplicity of the assay and the signal based on absorbance, the sensitivities of the assays were below dozens of picomolar concentrations of the analytes. In particular, the developed assay showed resistance to biological fluids upon the detection of analytes without complex pretreatment. The results demonstrate that the PPR platform is suitable and flexible for the development of simple and sensitive assay methods for detecting diverse molecules involved in important biological processes.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
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