荧光热漂移实验体外检测核小体的解聚

Fluorescence Thermal Shift Assay Detects Depolymerization of Nucleosomes in vitro

核小体是真核生物染色质的基本组成单元。核小体的解聚可以动态调控诸如DNA复制、转录、重组等以DNA为模板的生物学过程。特定荧光探针分子与蛋白质疏水残基结合后可被激发荧光信号。荧光热漂移(fluorescence thermal shift,FTS)是一种利用该原理检测温度上升时蛋白质变性过程的方法。本文以Widom 601序列为DNA模板,盐透析法体外装配核小体;分别在实时荧光定量PCR仪的VIC和TAMRA通道激发下,利用FTS检测了核小体在Tris-NaCl(TN)缓冲液和HEPES缓冲液中的解聚状态。研究结果发现,DNA对照序列在TN缓冲液中产生的背景噪声较大,VIC通道激发下组蛋白八聚体产生的荧光信号相对较强。FTS检测核小体的结果显示,随着温度的上升核小体的解聚过程分为2个阶段,~71℃和~84℃是核小体解聚速度最快的温度范围,而75℃~80℃是降解速度较慢的阶段。基于FTS基本原理,本研究发展了一种新颖的可用于检测核小体结构稳定性的方法,为核小体与染色质结构相关问题的研究奠定了一定的技术基础。

The nucleosome is the elementary repeating unit of chromatin in eukaryotes.Through adjusting the DNA accessibility of the regulatory proteins,nucleosome depolymerization can impact a lot of biological processes,such as transcription,DNA replication,DNA repair and so on.Fluorescence thermal shift(FTS)can monitor thermal unfolding of proteins at a critical temperature that depends upon its intrinsic stability in the presence of a fluorescent dye.A fluorescent probe that will fluoresce when bound to hydrophobic surfaces,is used to monitor protein unfolding as temperature is increased by using a real-time PCR instrument.In this work,nucleosomes were assembled by the salt-dialysis method on Widom-601 DNA templates.Nucleosome depolymerization in the TN and HEPES buffer were detected by FTS,and the fluorescence excitation channel of VIC and TAMRA in real-time PCR instruments was used in the FTS.The results showed that a large background noise of fluorescence signals can be detected while a probe monitored DNA control sample in TN buffers.The fluorescence signal is strong relatively in monitoring histone octamer samples through the VIC excitation channel.FTS assays showed that nucleosome depolymerization included two rapid dissociation stages,which occurred at^71℃and^84℃,respectively.Nucleosomes were slowly depolymerized between 75℃to 80℃.In conclusion,the FTS assay can be employed to detect nucleosome stability.The work might provide an effective experimental method for further study of nucleosome and chromatin structures.