생몰정보
소속
직위
직업
활동분야
주기
서지
국회도서관 서비스 이용에 대한 안내를 해드립니다.
검색결과 (전체 1건)
원문 있는 자료 (1) 열기
원문 아이콘이 없는 경우 국회도서관 방문 시 책자로 이용 가능
목차보기더보기
title page
Contents
Abbreviations 14
I. Overall Introduction 15
1. Programmed -1 ribosomal frameshifting (-1 FS) 15
2. RNA Pseudoknot 17
3. Successful applications of virtual screening against targeting RNA structure 21
3.1. Rational drug design and virtual screening 21
3.2. Successful applications of VS to discover ligands for RNA 22
II. Purpose of this study 24
Part 1: Identification of New Ligands for Biotin-Binding Pseudoknot by Structure-Based Virtual Screening 25
I. Introduction 26
1. Biotin-binding RNA pseudoknot 26
II. Materials and Methods 30
1. Structure-based virtual screening 30
1.1. Apparatus 30
1.2. Determination of pharmacophores on the basis of X-ray structure of the biotin-binding RNA pseudoknot 30
1.3. FlexX/CScore filtering 30
2. In vitro -1 frameshifting assay 33
2.1. Apparatus and materials 33
2.2. Template constructs for -1 frameshifting assay 34
2.3. In vitro transcription/translation coupled assay 37
2.4. Dual luciferase assay 38
2.5. In vitro transcription assay 38
2.6. In vitro translation assay 39
2.6.1. Synthesis of RNAs for in vitro translation assay 39
2.6.2. In vitro translation assay 40
3. Cell-based -1 frameshifting assay 41
3.1. Cell culture and transfection 41
3.2. Protein concentration estimation and dual luciferase assay 42
III. Results 44
1. Structure-based virtual screening. 44
1.1. Initial filtering of chemical database by 3D-pharmacophore search 44
1.2. Filtering by docking analyses using FlexX 44
1.3. Final docking with FlexX/CScoring 45
2. Screening of -1 frameshifting efficiency by in vitro assay 48
2.1. In vitro transcription/translation (TNT) coupled assay 48
2.2. In vitro dual luciferase assay 52
2.3. In vitro transcription assay 54
2.4. In vitro translation assay 56
3. Cell-based -1 frameshifting assay. 58
4. Selectivity of h4 toward the biotin-binding RNA pseudoknot 60
5. Docked model of h4 64
IV. Discussion 66
Part 2: Identification of Small Ligands that Regulate -1 Ribosomal Frameshifting in SARS-CoV 68
I. Introduction 69
1. Programmed -1 frameshifting in SARS-CoV 69
II. Materials and Methods 73
1. Structure-based design and virtual Screening 73
1.1. Apparatus 73
1.2. Modeling of RNA pseudoknot 73
1.2.1. Search for the site of-1 frameshifting site using Frameshifting Signal Finder (FSFinder) 73
1.2.2. Predict the structure of RNA Pseudoknot by PSEUDOVIEWER 3 76
1.2.3. Optimization of the 3D structure of pseudoknot using Amber 8.0 78
1.2.4. RNA receptor - small molecules docking methods : DOCK 78
2. In vitro -1 frameshifting assay 83
2.1. Apparatus and materials 83
2.2. Template constructs for -1 frameshifting assay 84
2.3. In vitro Transcription/Translation coupled assay 85
2.4. Dual Luciferase assay 88
2.5. In vitro transcription assay 88
2.6. In vitro translation assay 90
2.6.1. Synthesis RNA for in vitro translation assay 90
2.6.2. In vitro translation assay 90
3. Cell-based -1 frameshifting assay 91
3.1. Cell culture and transfection 91
3.2. Protein concentration estimation and dual luciferase assay 92
III. Results 94
1. Structure-based virtual screening 94
1.1. Modeling of the SARS-CoV RNA pseudoknot 94
1.2. Filtering of chemical database by DOCK 4.0 97
2. Screening of -1 frameshifting efficiency by in vitro assay 100
2.1. In vitro transcription/translation (TNT) coupled assay 100
2.2. In vitro dual luciferase assay 105
2.3. In vitro transcription assay 108
2.4. In vitro translation assay 110
3. Cell-based -1 frameshifting assay. 112
4. Selectivity of F7 toward the SARS-CoV RNA pseudoknot 116
5. Docked models of interesting compounds 124
IV. Discussion 127
V. Conclusion 129
VI. References 133
Abstract 144
국문 요약 146
Table 1. FlexX/CScore analysis. 47
Table 2. Fixed parameters for DOCK run 81
Table 3. Docking energy score analysis 99
Figure 1. Overview of a -1 frameshifing. 18
Figure 2. Schematic diagram of RNA secondary structure. 20
Part1. 9
Figure 1-1. Structure of the biotin-binding RNA pseudoknot. 27
Figure 1-2. Biotin binding site. 29
Figure 1-3. Vectors for the dual-luciferase reporter system 35
Figure 1-4. (A) Template construct for -1 frameshifting assay of the biotin-binding RNA pseudoknot. 36
Figure 1-5. Effects of candidate compounds on -1 frameshifting efficiencies in vitro TNT coupled assay. 49
Figure 1-6. A histogram of -1 frameshifting efficiencies obtained by SDS-PAGE analysis. 51
Figure 1-7. A histogram of the results obtained by dual luciferse assay indicating percent frameshift in vitro TNT coupled assay. 53
Figure 1-8. In vitro transcription with candidate compounds and the biotin-binding RNA pseudoknot. 55
Figure 1-9. -1 frameshifting efficiencies of a4, b2, d2 and e6 using in vitro translation assay. 57
Figure 1-10. -1 frameshifting efficiencies of hit compound h4 in cell-based experiment. 59
Figure 1-11. Template construct for -1 frameshifting of PEMV 61
Figure 1-12. -1 frameshifting efficiency of h4 on p21uc-PEMV system analyzed by SDS-PAGE. 62
Figure 1-13. -1 frameshifting efficiency of h4 on p21uc-PEMV system analyzed by dual luciferase assay 63
Figure 1-14. Docked model of compound h4 : biotin-binding RNA pseudoknot complex. 65
Part 2. 10
Figure 2-1. Genomic organization of the SARS-CoV and the mechanism of translation of open reading frame (ORF) 1b. 70
Figure 2-2. -1 Frameshifting site predicted by the program, FSFinder;http://wilab.inha.ac.kr/FSFinder/ 75
Figure 2-3. Structure data of the SARS-CoV RNA pseudoknot. 77
Figure 2-4. Template construct for -1 frameshifting. 87
Figure 2-5. Predicted model of 2D SARS-CoV RNA pseudoknt using PSEUDOVIEWER3 95
Figure 2-6. The 3D structure of the SARS-CoV pseudoknot based on PSEUDERVIEWER3. 96
Figure 2-7. -1 frameshifting efficiencies of compounds in vitro -1 frameshifting assay using SDS-PAGE. 101
Figure 2-8. -1 frameshifting efficiencies of compounds in vitro -1 frameshifting(frameshifitng) assay using SDS-PAGE. 103
Figrue 2-9. A histogram of-1 frameshifting efficiencies obtained by SDS-PAGE analysis. 104
Figure 2-10. A histogram of the results obtained by dual lucifcrse assay indicating percent -1 frameshifting. 107
Figure 2-11. In vitro transcription with candidate compounds and the SARS-CoV RNA pseudoknot. 109
Figure 2-12. -1 frameshifting efficiencies of F7, B3, and C6 using in vitro translation assay. 111
Figure 2-13. Effects of candidate compounds on -1 frameshifting efficiencies in cell-based experiments. 113
Figure 2-14. Concentration-dependent effects of F7 by HEK 293 T cell. 115
Figure 2-15. Template construct for -1 frameshifting of PEMV 117
Figure 2-16. -1 frameshifting efficiency of F7 on in vitro -1 frameshifting assay of PEMV analyzed by SDS-PAGE. 118
Figure 2-17. -1 frameshifting efficiency of F7 on in vitro -1 frameshifting assay of PEMV analyzed by dual luciferase assay. 120
Figure 2-18. -1 frameshifting efficiency of F7 on in vitro -1 frameshifting assay of p21uc-bio analyzed by SDS-PAGE. 122
Figure 2-19. -1 frameshifting efficiency of F7 on in vitro -1 frameshifting assay of p21uc-bio analyzed by dual luciferase assay. The reaction with only DMSO (lane1) considered as a control.... 123
Figure 2-20. Docked model of compound A6 : SARS-CoV RNA pseudoknot complex. 125
Figure 2-21. Docked model of compound F7 : SARS-CoV RNA pseudoknot complex. 126
Scheme 1-1. Overview of virtual screening strategy to discover novel ligands interacting with viral RNA pseudoknot 32
Scheme 1-2. Overview of -1 frameshifting assay in vitro and cell-based experiments. 43
Scheme 2-1. Overview of virtual screening strategy 82
Scheme 2-2. Overview of -1 frameshifing assay in vitro and cell-based experiments. 93
원문구축 및 2018년 이후 자료는 524호에서 직접 열람하십시요.
도서위치안내: / 서가번호:
우편복사 목록담기를 완료하였습니다.
* 표시는 필수사항 입니다.
* 주의: 국회도서관 이용자 모두에게 공유서재로 서비스 됩니다.
저장 되었습니다.
로그인을 하시려면 아이디와 비밀번호를 입력해주세요. 모바일 간편 열람증으로 입실한 경우 회원가입을 해야합니다.
공용 PC이므로 한번 더 로그인 해 주시기 바랍니다.
아이디 또는 비밀번호를 확인해주세요