Koopmans W J A, Brehm A, Logie C, Schmidt T, van Noort J
Physics of Life Processes, Leiden University, Leiden Institute of Physics, Niels Bohrweg 2, Leiden, The Netherlands.
J Fluoresc. 2007 Nov;17(6):785-95. doi: 10.1007/s10895-007-0218-9. Epub 2007 Jul 4.
We applied spFRET microscopy for direct observation of intranucleosomal DNA dynamics. Mononucleosomes, reconstituted with DNA containing a FRET pair at the dyad axis and exit of the nucleosome core particle, were immobilized through a 30 bp DNA tether on a polyethyleneglycol functionalized slide and visualized using Total Internal Reflection Fluorescence microscopy. FRET efficiency time-traces revealed two types of dynamics: acceptor blinking and intramolecular rearrangements. Both Cy5 and ATTO647N acceptor dyes showed severe blinking in a deoxygenated buffer in the presence of 2% betaME. Replacing the triplet quencher betaME with 1 mM Trolox eliminated most blinking effects. After suppression of blinking three subpopulations were observed: 90% appeared as dissociated complexes; the remaining 10% featured an average FRET efficiency in agreement with intact nucleosomes. In 97% of these intact nucleosomes no significant changes in FRET efficiency were observed in the experimentally accessible time window ranging from 10 ms to 10's of seconds. However, 3% of the intact nucleosomes showed intervals with reduced FRET efficiency, clearly distinct from blinking, with a lifetime of 120 ms. These fluctuations can unambiguously be attributed to DNA breathing. Our findings illustrate not only the merits but also typical caveats encountered in single-molecule FRET studies on complex biological systems.
我们应用单对荧光共振能量转移（spFRET）显微镜直接观察核小体内DNA的动态变化。单核小体由在二聚体轴和核小体核心颗粒出口处含有FRET对的DNA重构而成，通过一条30bp的DNA系链固定在聚乙二醇功能化载玻片上，并使用全内反射荧光显微镜进行观察。FRET效率随时间的变化曲线揭示了两种动态变化类型：受体闪烁和分子内重排。在含有2%β-巯基乙醇（betaME）的脱氧缓冲液中，Cy5和ATTO647N受体染料均表现出严重的闪烁现象。用1mM生育三烯酚（Trolox）取代三重态猝灭剂β-巯基乙醇消除了大部分闪烁效应。在抑制闪烁后，观察到三个亚群：90%表现为解离的复合物；其余10%的平均FRET效率与完整核小体一致。在97%的这些完整核小体中，在从10毫秒到数十秒的实验可及时间窗口内，未观察到FRET效率有显著变化。然而，3%的完整核小体显示出FRET效率降低的间隔，明显不同于闪烁，其寿命为120毫秒。这些波动可以明确归因于DNA呼吸。我们的研究结果不仅说明了单分子FRET研究在复杂生物系统中的优点，也说明了其典型的注意事项。
