Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes
Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes
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DOI：
10.1007/s00249-008-0383-z
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发表时间：
2009-04-01
影响因子：
2
通讯作者：
Bagshaw, Clive R.
中科院分区：
文献类型：
--
作者：
Cherny, Dmitry I.;Eperon, Ian C.;Bagshaw, Clive R.
单分子荧光显微镜是一种通过检测与生物大分子相关的荧光团产生的荧光信号来分析生物大分子动力学的方法。相邻的两个荧光团可能导致Forster共振能量转移(FRET)，这可以在单分子水平上检测到，并计算出能量转移的效率。在大多数情况下，实验观察到的FRET效率分布显示出相当大的宽度，对应于0.07-0.2(在0-1的范围内)。在这里，我们提出了一种通用的方法来描述DNA/RNA双链实验数据的分析。我们发现，对于Cy3和Cy5荧光团连接在螺旋两端的15BP双链，能量转移分布的宽度主要由光子散粒噪声和取向因子决定，而染料间距的变化起次要作用。
Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Forster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07-0.2 (on a scale of 0-1). Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.