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Title Page

ABSTRACT

Contents

General Introduction 22

Chapter I. Detection of cofilin mRNA by hybridization-sensitive fluorescent probes 27

I. Introduction 28

II. Results and Discussion 31

1. Design and synthesis of PyU-modified fluorescent probe for cofilin mRNA 31

2. Fluorescence enhancements of single strand probes 33

3. Quenching efficiencies and fluorescence enhancements of double-stranded probes 36

4. Formation of duplex structure 38

5. Thermal stability of double-stranded probe 40

6. Strand displacement of double-stranded probe 41

7. Selectivity of probe for single-base-mismatched target strands 43

III. Conclusion 44

IV. Experimental Section 45

1. Synthesis of oligonucleotides 45

2. Sample preparation for experiments 46

3. UV and fluorescence spectra 46

4. Melting temperatures (Tm)[이미지참조] 46

5. Circular dichroism (CD) spectroscopy 47

6. 20% Native polyacrylamide gel electrophoresis (PAGE) 47

7. MALDI-TOF mass results 48

V. References 49

Chapter II. Triplex-based PyA-modified guanine cluster for detection of AAG trinucleotide repeat[이미지참조] 51

I. Introduction 52

II. Results and Discussion 54

1. Minimum guanine bases of G-cluster for red-shifted fluorescence 54

2. Design of triplex-induced G-cluster probes for detection of AAG repeats 58

3. Optimization of triplex-induced G-cluster probes 59

4. G-cluster-induced triplex structures 62

5. Importance of G-cluster in triplex-forming probe 70

6. Sensitivity and selectivity of probes 72

7. Detection time of G-cluster triplex probe 76

8. Fluorescence changes for various long repeat sequences 77

9. Application to other purine-rich target strands 78

III. Conclusion 80

IV. Experimental Section 81

1. Synthesis of oligonucleotides 81

2. ODN sample preparation 82

3. UV and fluorescence spectra 82

4. Melting temperature (Tm)[이미지참조] 82

5. CD spectroscopy 83

6. Native polyacrylamide gel electrophoresis (PAGE) 83

7. MALDI-TOF mass results 84

V. References 85

Chapter III. G-cluster three-way junction probes for detection of various miRNAs 87

I. Introduction 88

II. Results and Discussion 89

1. Optimization of three-way junction probe design for detection of miR-21 89

2. Fluorescent properties of three-way junction probes for various target miRNAs 92

3. Formation of three-way junction structure 97

4. Response time and specificity of the probe for target miRNA 98

5. Imaging of miRNAs in cancer cells 100

III. Conclusion 104

IV. Experimental Section 105

1. Synthesis of oligonucleotides 105

2. ODN sample preparation 106

3. Fluorescence spectra 106

4. Melting temperature (Tm)[이미지참조] 106

5. 20% Polyacrylamide gel electrophoresis (PAGE) 107

6. Preparation of cells for confocal microscopy images 107

7. Confocal microscopy experiments 108

8. MALDI-TOF mass results 109

V. References 111

요약문 114

Abbreviations 117

Curriculum Vitae 118

List of Tables

Chapter I. Detection of cofilin mRNA by hybridization-sensitive fluorescent probes 20

Table 1. PyU-modified probe and target strands[이미지참조] 32

Table 2. Fluorescence enhancements of the probes for the target RNA 35

Table 3. Melting temperature (Tm) of each probe with its target RNA[이미지참조] 35

Table 4. Melting temperature of P1 in the presence of U5-U7 41

Table 5. MALDI-TOF mass 48

Chapter II. Triplex-based PyA-modified guanine cluster for detection of AAG trinucleotide repeat[이미지참조] 20

Table 1. Oligonucleotide sequences for the PyA-modified G-cluster[이미지참조] 55

Table 2. Triplex-forming G-cluster probes and target DNA AAG repeat sequences 60

Table 3. Fluorescence emission spectral analysis of the probes 62

Table 4. FAM-/BHQ-modified triplex-forming probe sequences 69

Table 5. Triplex-forming G-cluster probes and target DNA A-rich sequences 79

Table 6. Fluorescence emission spectral analysis of the probes for A-rich sequences 80

Table 7. MALDI-TOF mass 84

Chapter III. G-cluster three-way junction probes for detection of various miRNAs 21

Table 1. G-cluster three-way junction probe sequences with various number of the additional base pairs 90

Table 2. Spectral analysis of G-cluster three-way junction probes with various number of the additional base pairs 92

Table 3. G-cluster three-way junction probe sequences and target miRNA sequences 93

Table 4. Discrimination factors and Tm values of G-cluster three-way junction probes 96

Table 5. MALDI-TOF mass 109

List of Figures

Chapter I. Detection of cofilin mRNA by hybridization-sensitive fluorescent probes 12

Figure 1. Working principle of conventional molecular beacon (adapted from reference 1c) 28

Figure 2. (A) Conceptual representation of a QF-MB for RNA CAG trinucleotide repeats and (B) fluorescence image of solutions of QF-MB and its target (adapted from reference 5a) 29

Figure 3. Schematic representation of strand displacement process of double-stranded probe and (B) structure of fluorescent nucleoside PyU[이미지참조] 30

Figure 4. UV absorption spectra of probe strands (P1-P6) in the absence and presence of target RNA strands 34

Figure 5. Fluorescence emission spectra of probe strands (P1-P6) in the absence and presence of target RNA strands 34

Figure 6. Thermal melting curves of probe P1-P6 in the presence of target RNA 35

Figure 7. (A) Normalized UV absorption spectra, (B) fluorescence emission spectra and (C) relative fluorescence intensity of probe P1 with quencher strands (U5-U7) at 435... 37

Figure 8. Temperature-dependent UV absorption spectra of (A) P1, (B) P1+U5, and (C) P1+U6 38

Figure 9. Circular dichroism (CD) spectra of (A) P1 and T19 and (B-D) P1 in the presence of a quencher strand U5-U7 and T19 39

Figure 10. Native polyacrylamide gel electrophoresis (PAGE) images of P1 with T19 in the presence of U5-U7 (A) stained with Stains-All and (B) under long wavelength UV irradiation 40

Figure 11. Time-dependent fluorescence intensity of P1 in the presence of U5-U7 after the addition of T19 42

Figure 12. Fluorescence emission spectra of P1 in the presence of single-base-mismatched target RNA; 1.0 ㎛ of sample in 100 mM Tris-HCl buffer (pH 7.2), 100... 43

Chapter II. Triplex-based PyA-modified guanine cluster for detection of AAG trinucleotide repeat[이미지참조] 13

Figure 1. (A) Base interactions in parallel triplex and (B) the parallel triplex structure containing AAG repeat and chemical structures of parallel T-AT and C+-GC triplets... 53

Figure 2. (A) Fluorescence emission spectra and (B) photograph of solutions of the GC1+GC2, GC3+GC4, and GC5+GC6 duplexes 56

Figure 3. UV/Vis absorption spectra of (A) GC1+GC2, (B) GC3+GC4, and (C) GC5+GC6 duplexes 56

Figure 4. Thermal melting curves and Tm values of GC1+GC2, GC3+GC4, and GC5+GC6 duplexes[이미지참조] 57

Figure 5. Circular dichroism (CD) spectra of (A) GC1+GC2, (B) GC3+GC4, and (C) GC5+GC6 57

Figure 6. 20% Native polyacrylamide gel electrophoresis (PAGE) images of GC1-GC6 (A) stained with Stains-All and (B) under long wavelength UV irradiation. Lane 1: GC1;... 57

Figure 7. Schematic representation of a triplex-induced G-cluster probe for AAG repeats 59

Figure 8. Fluorescence emission spectra of triplex probes featuring various numbers of base pairs, recorded in the absence and presence of d(AAG)₄. (A) T4G0a+T4G0b, (B)... 61

Figure 9. Fluorescence emission spectra of T4G4a+T4G4b recorded at acidic and neutral pH. Buffer: 10 mM sodium phosphate, 10 mM MgCl₂. 63

Figure 10. 15% Native PAGE images of T4G4a and T4G4b (A) stained with Stains-All and (B) under long wavelength UV irradiation. Lane 1: T4G4a; lane 2: T4G4b; lane 3:... 63

Figure 11. 15% Native PAGE images of T4G4a and T4G4b (A) stained with Stains-All and (B) under long wavelength UV irradiation. Lane 1: T4G4a; lane 2: T4G4b; lane 3:... 64

Figure 12. Fluorescence emission spectra of T4G4a-m+T4G4b-m in the absence and presence of AAG4-m 65

Figure 13. 15% Native PAGE images of T4G4a-m and T4G4b-m (A) stained with Stains-All and (B) under long wavelength UV irradiation. Lane 1: T4G4a-m; lane 2:... 66

Figure 14. 15% Native PAGE images of T4G4a-m and T4G4b-m (A) stained with Stains-All and (B) under long wavelength UV irradiation. Lane 1: T4G4a-m; lane 2:... 66

Figure 15. CD spectra of T4G4a+T4G4b recorded in the presence and absence of d(AAG)₄ 67

Figure 16. (A) Schematic representation, (B) fluorescence emission spectra and (C) CD spectra of the hairpin-type probe T4G4H 68

Figure 17. 15% Native PAGE images of T4G4H and T4G4b (A) stained with Stains-All and (B) under long wavelength UV irradiation. Lane 1: T4G4H; lane 2: T4G4b; lane 3:... 68

Figure 18. (A) Schematic representation of the FAM-/BHQ-modified triplex-forming probe; (B) fluorescence emission spectra of T4N4a-F+T4N4b-Q in the absence and... 70

Figure 19. 15% Native PAGE images of T4N4a-F and T4N4b-Q (A) stained with Stains-All and (B) under long wavelength UV irradiation. Lane 1: T4N4a-F; lane 2: T4N4b-Q;... 70

Figure 20. Fluorescence emission spectra of T4A4a+T4A4b recorded in the absence and presence of d(AAG)₄. Excitation wavelength: 385 ㎚; Ex./Em. slit: 2.5/5 ㎚. 71

Figure 21. 15% Native PAGE images of T4A4a and T4A4b (A) stained with Stains-All and (B) under long wavelength UV irradiation. Lane 1: T4A4a; lane 2: T4A4b; lane 3:... 72

Figure 22. Concentration-dependent fluorescence emission spectra and analyses of (A-C) T4G4a+T4G4b and (D-F) T4G4a-m+T4G4b-m. Sample concentration: 100 nM;... 73

Figure 23. Selectivity of the triplex-forming probe T4G4a+T4G4b toward various repeat and random sequences. (A) Fluorescence emission spectra and (B) fluorescence intensity... 74

Figure 24. Selectivity of the probe T4G4a+T4G4b toward the excess amount of various repeat and random sequences. (A) Fluorescence emission spectra; (B) fluorescence... 75

Figure 25. Fluorescence emission spectra of T4G4a+T4G4b recorded in the absence and presence of repeat and random sequence mixture. Sample concentration: 1.5 μM of... 75

Figure 26. Concentration-dependent fluorescence emission spectra and analysis of T4G4a+T4G4b in response to d(AAG)₄. Sample concentration: 100 nM of T4G4a and... 76

Figure 27. Time-dependent fluorescence intensity changes of T4G4a+T4G4b at 580 ㎚ recorded in the absence and presence of d(AAG)₄. Temperature: 25 °C; time interval: 5 min. 77

Figure 28. Fluorescence emission spectra of T4G4a+T4G4b toward long repeat sequence (A) d(AAG)8 (0.5 eq.), (B) d(AAG)₁₂ (0.33 eq.), (C) d(AAG)16 (0.25 eq.), (D) d(AAG)20...[이미지참조] 78

Figure 29. Fluorescence emission spectra of triplex-induced G-cluster probes for A-rich sequences 79

Chapter III. G-cluster three-way junction probes for detection of various miRNAs 18

Figure 1. Schematic representation of design strategy for G-cluster three-way junction probe 90

Figure 2. Fluorescence emission spectra of G-cluster three-way junction probes for miR-21 91

Figure 3. Fluorescence emission spectra of G-cluster three-way junction probes for various target miRNAs 95

Figure 4. Fluorescence intensity ratios (I580/I455) of G-cluster three-way junction probes for various target miRNAs 96

Figure 5. 20% PAGE image of S4a-191+S4b-191 and S4a-181a+S4b-181a in the absence and presence of each target miRNA compared to natural three-way junction... 97

Figure 6. Concentration-dependent fluorescence spectra of (A) S4a+S4b and (b) S4a+S4b+miR-21 by changing the concentration of MeCl₂ from 0 mM to 50 mM. (C)... 98

Figure 7. Time-dependent fluorescence spectra of (A) S4a+S4b, (B) S4a-191+S4b-191, and (C) S4a-181a+S4b-181a after addition of each target miRNAs; miR-21, miR-191,... 99

Figure 8. Fluorescence ratios (I580/I455) of (A) S4a+S4b, (B) S4a-191+S4b-191 and (C) S4a-181a+S4b-181a for various miRNAs[이미지참조] 100

Figure 9. Concentration-dependent fluorescence emission spectra of probe for (A) miR-21, (B) miR-191, and (C) miR-181a with various target concentration. Sample... 100

Figure 10. Confocal microscopy images for cancer cell lines (HeLa, MCF-7, and HepG2) and normal cell line (HEK-293) treated with S4a+S4b. Fluorescence images obtained... 102

Figure 11. Confocal microscopy images for cancer cell lines (HeLa, MCF-7, and HepG2) and normal cell line (HEK-293) treated with S4a-191+S4b-191. Fluorescence images... 103

초록보기

 핵산 구조에 기반한 형광 프로브 시스템은 서열에 따라 예측 가능한 이차구조를 이룰 수 있으며, 생물학적으로 중요한 핵산들을 탐지하기 위한 수단으로 사용되어 왔다. 핵산 시스템에서 핵산의 서열과 형광체의 도입 위치를 조절함으로써 핵산에 도입된 형광체 간 또는 형광체와 다른 분자 간 상호작용을 조절하여 의도된 광물리적 및 구조적 특성을 유도하는 것이 가능하다. 본 연구에서는 반복 서열을 가진 DNA, miRNA, mRNA와 같은 핵산들을 탐지하기 위하여, 높은 양자수율과 핵산의 구조에 따라 특징적인 형광을 발산하는 파이렌이 도입된 뉴클레오시드를 이용하였으며, 화학적으로 변형된 뉴클레오시드를 다양한 구조의 핵산에 도입하여 (1) 코필린 mRNA의 3'-UTR, (2) AAG 삼염기 반복서열, (3) 다양한 miRNA를 탐지할 수 있는 새로운 형광 핵산 시스템을 개발하였다.

Chapter I. Hybridization에 민감한 형광 프로브를 이용한 코필린 mRNA의 탐지

주변 환경에 민감한 형광 뉴클레오시드인 PyU를 프로브 서열에 도입하여 신경세포에서 신호 전달에 관련된 코필린 mRNA를 탐지하는 형광 핵산 프로브 시스템을 개발하였다. 형광체가 도입된 프로브의 배경 신호를 낮추기 위하여 PyU 간의 상호작용을 통해 높은 소광 효과를 얻을 수 있는 부분 이중나선 구조를 적용하였다. 이전의 소광체가 없는 분자 비컨은 특정 반복서열에만 적용이 가능하였지만, 본 연구에서 개발된 형광 핵산 프로브는 부분 이중나선 구조의 도입을 통해 다양한 서열을 갖는 표적 RNA에도 적용될 수 있으며, 부분 이중나선 프로브와 표적 RNA와의 서열 재배치를 통해 높은 형광 향상을 초래한다.

Chapter II. AAG 삼염기 반복서열 탐지를 위한 삼중나선에 기반한 PyA-변형 구아닌 클러스터

삼중나선과 PyA-변형 구아닌 클러스터 (G-클러스터)의 조합은 DNA AAG 반복서열을 검출하기 위한 새로운 형광 핵산 프로브 시스템을 제시하였다. AAG 반복서열과 이에 상보적인 서열로 구성되며 중성 pH에서 특이적으로 안정화되는 삼중나선 구조를 프로브 구조로 선택하였다. PyA과 이웃한 구아닌 염기들로 구성된 G-클러스터는 A-클러스터보다 더 높은 열역학적 안정성과 향상된 장파장 형광 방출을 보였다. 이 시스템에서는 표적 AAG 반복서열과의 결합을 통해 극적인 형광 색 변화를 유도하기 위하여 G-클러스터를 형광 단위체로 사용하였다. 삼중나선 구조를 형성하는 G-클러스터 프로브는 다양한 반복 서열에 대하여 높은 선택성을 보였으며, 프로브의 형광 신호는 AAG 반복 서열에 대하여 매우 민감한 형광 변화를 보였다. 또한, 긴 AAG 반복서열에 대해서도 높은 장파장 형광을 방출하며 FRDA를 유발하는 DNA AAG 반복서열의 탐지 가능성을 보였다.

Chapter III. 다양한 miRNA 탐지를 위한 G-클러스터 three-way junction 프로브

본 연구에서는 다양한 miRNA의 탐지와 시각화를 위하여 three-way junction 프로브 시스템에 G-클러스터를 적용하였다. PyA와 구아닌 염기로 구성된 G-클러스터는 표적 miRNA와 three-way junction 구조를 형성하며 강한 장파장 형광 발산을 유도하였다. 다양한 miRNA 서열에 대하여 G-클러스터 three-way junction 시스템을 적용하였으며, 프로브는 각각의 표적 miRNA와의 결합하여 three-way junction 구조를 형성함으로써 높은 형광 파장 변화를 보였다. 세 종류의 암 세포 (HeLa, MCE-f, HepG2)에 three-way junction 프로브를 적용하여 암 세포에서 발현되는 표적 miRNA를 감지하고 시각화하여, G-클러스터 탐지 시스템이 세포 내 다양한 miRNA를 탐지할 수 있음을 확인하였다.

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