1)清华大学生命科学学院,北京100084;2)清华大学蛋白质研究技术中心,北京100084
Tel:
国家自然科学基金(22007054)和清华大学实验室创新基金资助项目.
1)School of Life Sciences, Tsinghua University, Beijing 100084, China;2)Technology Center for Protein Sciences, Tsinghua University, Beijing 100084, China
This work was supported by grants from The National Natural Science Foundation of China (22007054) and Innovation Funding from Office of Laboratory Management, Tsinghua University.
荧光寿命是指荧光分子在回到基态前在激发态停留的平均时间. 本文发展了基于荧光寿命测量来定量分子内和分子间相互作用的方法:通过G碱基猝灭对于荧光寿命的影响定量DNA二级结构的形成;通过荧光共振能量传递(FRET)中荧光寿命的变化来定量分子间的相互作用. 第一种方法巧妙利用了G碱基会猝灭临近的染料分子的性质,结合荧光寿命的变化,可以判断DNA二级结构的形成以及形成的比率. FRET是用于研究生物分子相互作用的一个重要手段. 传统的FRET方法主要是基于强度的变化,但这种变化容易受到荧光表达水平变动、样品中分子扩散以及荧光串色的影响,因此经常存在着实验比较复杂和重复性差的问题. 而基于荧光寿命的FRET研究则可以很好地克服上述缺点. 通过检测供体荧光寿命的变化,我们能够方便快捷地判断是否发生FRET,并通过建立系统的数据分析方法,得到FRET的效率以及分子之间相互作用的信息.
Fluorescence lifetime is the average amount time a fluorophore spends in excited state before returning to the ground state. In this paper, methods for quantifying intra- and inter-molecular interactions based on fluorescence lifetime measurement were developed. One is quantification of DNA secondary structures formation by fluorescence lifetime change through G-base quenching, and the other is quantification of inter-molecular interactions by fluorescence lifetime change through fluorescence resonance energy transfer (FRET). The first method cleverly utilizes the properties of G-base to quench adjacent dye molecules, and combines with changes in fluorescence lifetime, to determine the formation of DNA secondary structure and the formation ratio. FRET is an important means for studying biological molecular interactions. Traditional FRET methods are mainly based on change in intensity, but this change is susceptible to changes in fluorescence expression levels, molecular diffusion in samples and crosstalk between fluorophores, which brings complexity in experimental design and poor repeatability of experimental data. FRET measurement based on fluorescence lifetime can overcome the disadvantages mentioned above. By detecting changes in the fluorescence lifetime of the donor, we can quickly and easily determine whether FRET occurs, and through establishing a systematic data analysis method we can get FRET efficiency and information about inter-molecular interactions.
王文娟.利用荧光寿命变化定量分子内和分子间相互作用的方法[J].生物化学与生物物理进展,2021,48(11):1358-1364
复制生物化学与生物物理进展 ® 2025 版权所有 ICP:京ICP备05023138号-1 京公网安备 11010502031771号