利用荧光寿命变化定量分子内和分子间相互作用的方法

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...

Quantification of Intra- and Inter-molecular Interactions Based on Fluorescence Lifetime Measurement

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.