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Table of Contents
Intro
Supervisor's Foreword
Abstract
Acknowledgements
Contents
List of Figures
List of Tables
List of Schemes
1 Introduction
1.1 Protein-RNA Interaction
1.2 Therapeutic Potentials of Modulating PRI
1.3 Chemical Biology Approaches for the Discovery of PRI Modulator
1.4 Aims and Scope of the Thesis
References
2 Identification of Small-Molecule Inhibitors of Oncogenic Lin28-Let-7 Interaction
2.1 Discovery of a Small-Molecule Inhibitor of Protein-microRNA Interaction Using Binding Assay with a Site-Specifically Labeled Lin28
2.1.1 Introduction
2.1.2 Results and Discussion
2.1.3 Conclusion
2.1.4 Experimental Section
2.2 Restoring Let-7 MicroRNA Biogenesis Using a Small-Molecule Inhibitor of the Protein-RNA Interaction
2.2.1 Introduction
2.2.2 Results and Discussion
2.2.3 Conclusion
2.2.4 Experimental Section
References
3 Discovery of Small-Molecule Modulators of Protein-RNA Interactions by Fluorescence Intensity-Based Binding Assay
3.1 Introduction
3.2 Results and Discussion
3.2.1 Construction of a Fluorescence Intensity-Based Lin28-Let-7 Binding Assay
3.2.2 Discovery of a New Class of Small-Molecule Inhibitors of the Lin28-Let-7 Interaction
3.2.3 Construction of a Fluorescence Intensity-Based Assay to Monitor Roquin-Tnf CDE Interaction
3.3 Conclusion
3.4 Experimental Section
3.4.1 Protein Expression and Purification
3.4.2 Fluorescence Intensity-Based Binding Assays
3.4.3 Electrophoretic Mobility Shift Assay (EMSA)
3.4.4 RNA Sequences
3.4.5 TaqMan MiRNA Assay-Based QRT-PCR
3.4.6 Cell Culture
3.4.7 Western Blot
3.4.8 Chemistry
3.4.9 Purity Determination from HPLC Analysis
3.4.10 Synthetic Procedures and Spectroscopic Data
References
4 Harnessing Stress Granule Formation by Small Molecules to Inhibit the Cellular Replication of SARS-CoV-2
4.1 Introduction
4.2 Results and Discussion
4.2.1 Identification of Small-Molecule Enhancers of Stress Granule Formation and Their Cellular Antiviral Activities
4.2.2 Inhibition of the Cellular Replication of SARS-CoV-2 by the Stress Granule Enhancers
4.3 Conclusion
4.4 Experimental Section
4.4.1 Chemistry
4.4.2 Purity Determination from HPLC Analysis
4.4.3 Cell Culture and Transfection
4.4.4 Western Blot
4.4.5 Stress Granule Imaging by Immunofluorescence
4.4.6 Stress Granule Monitoring and Compound Screening Using G3BP1-GFP Expressed U2OS Cell Line
4.4.7 Cell Viability Assay
4.4.8 RNA Extraction and Quantitative Real-Time PCR
4.4.9 Virus
4.4.10 Antiviral Test and Dose-response Curve (DRC) Analysis by Immunofluorescence
4.4.11 Co-Immunofluorescence
4.4.12 Drug Combination Assay
4.4.13 Spectroscopic, Mass & HPLC Purity Data
References
Appendix
Supervisor's Foreword
Abstract
Acknowledgements
Contents
List of Figures
List of Tables
List of Schemes
1 Introduction
1.1 Protein-RNA Interaction
1.2 Therapeutic Potentials of Modulating PRI
1.3 Chemical Biology Approaches for the Discovery of PRI Modulator
1.4 Aims and Scope of the Thesis
References
2 Identification of Small-Molecule Inhibitors of Oncogenic Lin28-Let-7 Interaction
2.1 Discovery of a Small-Molecule Inhibitor of Protein-microRNA Interaction Using Binding Assay with a Site-Specifically Labeled Lin28
2.1.1 Introduction
2.1.2 Results and Discussion
2.1.3 Conclusion
2.1.4 Experimental Section
2.2 Restoring Let-7 MicroRNA Biogenesis Using a Small-Molecule Inhibitor of the Protein-RNA Interaction
2.2.1 Introduction
2.2.2 Results and Discussion
2.2.3 Conclusion
2.2.4 Experimental Section
References
3 Discovery of Small-Molecule Modulators of Protein-RNA Interactions by Fluorescence Intensity-Based Binding Assay
3.1 Introduction
3.2 Results and Discussion
3.2.1 Construction of a Fluorescence Intensity-Based Lin28-Let-7 Binding Assay
3.2.2 Discovery of a New Class of Small-Molecule Inhibitors of the Lin28-Let-7 Interaction
3.2.3 Construction of a Fluorescence Intensity-Based Assay to Monitor Roquin-Tnf CDE Interaction
3.3 Conclusion
3.4 Experimental Section
3.4.1 Protein Expression and Purification
3.4.2 Fluorescence Intensity-Based Binding Assays
3.4.3 Electrophoretic Mobility Shift Assay (EMSA)
3.4.4 RNA Sequences
3.4.5 TaqMan MiRNA Assay-Based QRT-PCR
3.4.6 Cell Culture
3.4.7 Western Blot
3.4.8 Chemistry
3.4.9 Purity Determination from HPLC Analysis
3.4.10 Synthetic Procedures and Spectroscopic Data
References
4 Harnessing Stress Granule Formation by Small Molecules to Inhibit the Cellular Replication of SARS-CoV-2
4.1 Introduction
4.2 Results and Discussion
4.2.1 Identification of Small-Molecule Enhancers of Stress Granule Formation and Their Cellular Antiviral Activities
4.2.2 Inhibition of the Cellular Replication of SARS-CoV-2 by the Stress Granule Enhancers
4.3 Conclusion
4.4 Experimental Section
4.4.1 Chemistry
4.4.2 Purity Determination from HPLC Analysis
4.4.3 Cell Culture and Transfection
4.4.4 Western Blot
4.4.5 Stress Granule Imaging by Immunofluorescence
4.4.6 Stress Granule Monitoring and Compound Screening Using G3BP1-GFP Expressed U2OS Cell Line
4.4.7 Cell Viability Assay
4.4.8 RNA Extraction and Quantitative Real-Time PCR
4.4.9 Virus
4.4.10 Antiviral Test and Dose-response Curve (DRC) Analysis by Immunofluorescence
4.4.11 Co-Immunofluorescence
4.4.12 Drug Combination Assay
4.4.13 Spectroscopic, Mass & HPLC Purity Data
References
Appendix