研究目的
To develop a fast and efficient method for fluorogenic labeling and imaging of DNA in vitro and in vivo using coumarin-fused tetrazoles under UV LED photoirradiation.
研究成果
The study successfully demonstrated the fast fluorogenic labeling and imaging of DNA in vitro and in vivo using coumarin-fused tetrazoles. The water-soluble, nuclear-specific coumarin-fused tetrazole (CTz-SO3) enabled effective labeling and visualization of metabolically synthesized DNA in cultured cells and live zebrafish, offering a new tool for spatiotemporal-controlled investigation of cellular processes involving nucleic acids.
研究不足
The study is limited by the use of UV light for photoirradiation, which may not be suitable for all biological applications due to potential cellular damage. The fluorogenic probes may also have limited applicability in systems with high background fluorescence.
1：Experimental Design and Method Selection:
The study involved the design and synthesis of coumarin-fused tetrazoles for photoactivated fluorogenic labeling of DNA. The methodology included UV-VIS absorption and emission spectra analysis, density functional theory calculations, and confocal fluorescence microscopy for imaging.
2：Sample Selection and Data Sources:
Cultured cells (A549, U87, EMT6, and 4T1) and zebrafish embryos were used as biological samples. DNA sequences modified with acrylamide by an alkyl linker at the 5’ end were synthesized for in vitro studies.
3：List of Experimental Equipment and Materials:
Equipment included a ZEISS LSM 510 META confocal microscope, UV LED photoirradiation setup, and HPLC for analysis. Materials included coumarin-fused tetrazoles, VdU, and other chemical reagents.
4：Experimental Procedures and Operational Workflow:
The procedure involved treating cells with VdU, incubating with tetrazoles, photoirradiation, and fluorescence imaging. Zebrafish embryos were injected with VdU, incubated with tetrazoles, and imaged after photoirradiation.
5：Data Analysis Methods:
Data analysis included fluorescence and absorption spectra examination, PAGE analysis, and confocal microscopy image processing.
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