Fluorescent Protein : Properties and Applications in Biology
- 주제어 (키워드) Fluorescent Protein , Fluorescence intensity , Photostability , DNA staining , DNA labeling
- 발행기관 서강대학교 일반대학원
- 지도교수 조규봉
- 발행년도 2025
- 학위수여년월 2025. 2
- 학위명 박사
- 학과 및 전공 일반대학원 화학과
- 실제 URI http://www.dcollection.net/handler/sogang/000000079628
- UCI I804:11029-000000079628
- 본문언어 영어
- 저작권 서강대학교 논문은 저작권 보호를 받습니다.
초록 (요약문)
This thesis presents a comprehensive study on fluorescent protein (FP)- based DNA visualization techniques, with a focus on developing FP-DNA binding proteins for single-molecule imaging applications. The first part of this work details the engineering of 21 types of fluorescent protein-DNA binding proteins (FP-DBPs), created by combining various FPs with high mobility group (HMG) and truncated transcription activator-like effector (tTALE) DNA-binding motifs. This diverse array of FP-DBPs facilitated DNA-specific staining across multiple wavelengths and brightness levels, significantly broadening the functional capabilities of DNA imaging tools. The effects of DBP type and DBP coupling orientation on fluorescent brightness were investigated. Additionally, DNA staining patterns were examined using tTALE as the DBP and varying salt concentrations, yielding insights into the specificity and brightness of FP-DBPs. The second part of this study evaluates the fluorescence intensity of the StayGOLD FP (SA-StayGOLD) for DNA labeling applications, specifically in comparison to mNeonGreen. The results demonstrate that SA-StayGOLD provides superior fluorescence intensity, largely due to its enhanced photostability, establishing it as an optimal choice for high-resolution, long-duration imaging. The study also suggests that specific protein-chromophore interactions contribute to the photostability of StayGOLD. This research advances FP-based DNA visualization through the exploration of various FP combinations. These tools provide a foundation for single-molecule studies, which we aim to apply to the optical mapping of genomic DNA.
more목차
Chapter 1 Introduction 1
1.1 DNA Staining and Labeling with Organic Dye 1
1.2 Fluorescent proteins and their properties 2
1.3 Fluorescent protein-based staining and labeling of DNA 5
Chapter 2 Twenty-one Fluorescent Protein-Based DNA Staining Dyes 7
2.1 Methods 7
2.1.1 Chemicals 7
2.1.2 FP-DBP cloning 7
2.1.3 DNA Imaging on a Positively Charged Surface and Fluorescence Intensity Measurement 11
2.1.4 DNA Imaging on a Positively Charged Surface with Microchannels 12
2.1.5 Fluorescence Intensity Measurement with the Fluorometer 12
2.2 An Overview 13
2.3 Results and Discussion 13
2.3.1 Construction and Expression of Diverse HMG-FPs and tTALE-FPs 13
2.3.2 Brightness Optimization of FP-DBPs 16
2.3.3 A/T-Rich Specific Staining by tTALE-FP 20
Chapter 3 Fluorescent Properties and Photostability of Streptavidin-Coupled StayGOLD Protein for DNA Labeling 25
3.1 Methods 25
3.1.1 Chemicals 25
3.1.2 Construction, Expression, and Purification of SA-FPs 27
3.1.3 Sequence-specific Biotin-Labeling of λ DNA 28
3.1.4 Characterization of SA-FPs 28
3.2 An Overview 30
3.3 Results and Discussion 30
3.3.1 Integrated intensities of labeled SA-FPs 30
3.3.2 Photophysical and Structural Analysis of SA-FPs 36
Chapter 4 Applications using Fluorescent Protein 41
4.1 Introduction 41
4.2 FP application in Other DNA studies 42
4.3 FP application in Cell Biology 44
4.4 Conclusion 48
Appendix 50
References 81
