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Development of a molecular sensing platform technology based on proximity proteolysis reaction

초록/요약

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.

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목차

CHAPTER 1. General introduction 1
1.1 Biological marker 2
1.2 Conventional biomarker sensing methods 4
1.3 Protein switch 9
1.4 Molecular proximity and its application for biosensing 15
1.5 Protein-DNA conjugates 17
1.6 Aims of this study 23
CHAPTER 2. Nucleic acid detection by a proximity proteolysis reaction 26
2.1 Abstract 27
2.2 Introduction 28
2.3 Materials and Methods 30
2.3.1 Plasmid construction for expressing proteins 30
2.3.2 Protein expression and purification 30
2.3.3 Derivatization of ssDNA with N-hydroxysuccimide ester-(polyethyleneglycol)4-dibenzylcyclooctyne (NHS-PEG4-DBCO) 32
2.3.4 Conjugation of single-stranded DNA (ssDNA) and protein 32
2.3.5 Determination of protein and DNA concentration for the ssDNA-protein conjugates 33
2.3.6 Enzyme activity assay for β-lactamase zymogen 33
2.3.7 Enzyme activity assay for TEV protease 34
2.3.8 Nucleic acid detection by proximity proteolysis reaction 34
2.3.9 Nucleic acid sequence-based amplification (NASBA) 35
2.4 Results and Discussion 38
2.4.1 Choice of a pair of protease and zymogen 38
2.4.2 Conjugation of β-lactamase zymogen and single-stranded DNA (ssDNA) 39
2.4.3 Conjugation of TEV protease and ssDNA 43
2.4.4 Set-up of proximity proteolysis reaction 46
2.4.5 Detection of DNA and RNA 50
2.4.6 Combination of proximity protease reaction with nucleic acid sequence-based amplification (NASBA) 55
2.5 Conclusion 57
CHAPTER 3. Design of hairpin-loop structured DNA for a universal detection of RNA 58
3.1 Abstract 59
3.2 Introduction 60
3.3 Materials and Methods 63
3.3.1 Simulation via nucleic acid package (NUPACK) 63
3.3.2 Dynamic binding analysis 63
3.3.3 PPR assay based on HP probe 63
3.3.4 Cell lysate preparation 64
3.3.5 In vitro transcription 64
3.4 Results and Discussion 65
3.4.1 Principle of HP probe based PPR to detect nucleic acids 65
3.4.2 Design of HP construct for universal probe 67
3.4.3 Selection of length of ssDNA to optimize the reversible binding of zymogen-ssDNA conjugates 70
3.4.4 Detection of synthetic DNA with identical miRNA sequence 79
3.4.5 miRNA21 detection in crude cell lysates 86
3.4.6 Applying the assay for mRNA detection 88
3.5 Conclusion 91
CHAPTER 4. Homogeneous immunoassay by a proximity proteolysis reaction 92
4.1 Abstract 93
4.2 Introduction 94
4.3 Materials and Methods 96
4.3.1 Antibody and human chorionic gonadotropin (hCG) production by mammalian cells 96
4.3.2 Single-stranded DNA (ssDNA)-binder conjugation and purification 96
4.3.3 Conjugation of ssDNA and ε-(digoxigenin-3-0-acetamido)caproic acid N-hydroxysuccinimide ester (digoxigenin-NHS ester) 97
4.3.4 PPR-based immunoassay 97
4.3.5 Mammalian cell preparation 97
4.3.6 Flow cytometry analysis for the confirmation of HER2 expression level 98
4.4 Results and Discussion 99
4.4.1 Design of PPR-based immunoassay 99
4.4.2 Proof of PPR-based immunoassay 101
4.4.3 Detection of proteins using the PPR-based immunoassay 106
4.4.4 Detection of HER2 on the cell membrane 110
4.4.5 Detection of thrombin using aptamer and validation of assay specificity 114
4.4.6 Antibody sensing based-on antigen-ssDNA conjugate 120
4.4.7 Small molecule sensing using competitive PPR-based immunoassays 126
4.5 Conclusion 129
CHAPTER 5. Summary 131
References 135
Abstract in Korean 149

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