Title Page
Contents
ABSTRACT 9
Chapter 1. Introduction 11
1.1. The potential risks associated with COVID-19 11
1.2. Life cycle of SARS-CoV-2 and conservation between coronaviruses NSP13 13
1.3. Function and Structure of NSP13 18
1.4. Mechanism of NSP13 20
1.5. Function and Structure of NSP13 with RTC 22
Chapter 2. Materials and Methods 24
2.1. Reagents 24
2.2. Cloning and expression of NSP13 24
2.3. Purification 24
2.4. FRET-based assay 25
2.5. Accuracy test of FRET-based assay 29
Chapter 3. Results and Discussions 31
3.1. NSP13 structure between coronaviruses 31
3.2. NSP13 activity between coronaviruses 35
3.3. Purification 37
3.4. FRET-based enzyme assay 44
3.5. FRET-based assay accuracy test 46
3.6. Enzyme kinetics of NSP13 48
3.6.1. NSP13 concentration-dependent unwinding activity 48
3.6.2. DNA concentration-dependent unwinding activity 50
3.6.3. ATP concentration-dependent unwinding activity 52
3.7. NSP13 inhibitor between coronaviruses 54
Chapter 4. Conclusion 57
References 59
Abstract (in Korean) 63
Table 1. Comparison between coronaviruses 12
Table 2. Sequence conservation between coronaviruses NSP13 17
Table 3. The DNA sequence of FRET-based assay 28
Table 4. The DNA sequence of the FRET-based assay accuracy test 30
Table 5. NSP13 structure between coronaviruses 33
Table 6. NSP13 activity between coronaviruses 36
Table 7. Inhibitors of NSP13 between coronaviruses 56
Figure 1. The life cycle of SARS-CoV-2 14
Figure 2. Sequence alignment between coronaviruses NSP13 16
Figure 3. Overall Structure of NSP13(PDB ID: 7NIO) 19
Figure 4. Mechanism of NSP13 unwinding activity 21
Figure 5. Overall Structure of nsp13₍₂₎-RTC (PDB ID: 7RE1) 23
Figure 6. Principle of the FRET-based assay 26
Figure 7. Structural differences between SARS-CoV NSP13(PDB ID: 6JYT) and SARS-CoV-2 NSP13(PDB ID: 7NIO) 32
Figure 8. Structural differences between MERS-CoV nsp13(PDB ID: 5WWP) and SARS-CoV-2 nsp13 34
Figure 9. The flow of purification 38
Figure 10. SDS-PAGE analysis of Ni-NTA chromatography 39
Figure 11. SDS-PAGE analysis of cation exchange chromatography 40
Figure 12. SDS-PAGE analysis of TEV cleavage site cut and 2nd Ni-NTA chromatography[이미지참조] 41
Figure 13. SDS-PAGE analysis of size exclusion chromatography 42
Figure 14. DNA/RNA free form NSP13 43
Figure 15. Principle of FRET-based assay on Hairpin/Non-hairpin DNA 45
Figure 16. FRET-based assay accuracy test 47
Figure 17. Initial activity dependent on NSP13 concentration 49
Figure 18. Initial activity dependent on DNA concentration 51
Figure 19. Initial activity dependent on ATP concentration 53
Figure 20. Inhibitors of SARS-CoV-2 NSP13 55