Cherny Dmitry I, Eperon Ian C, Bagshaw Clive R
Department of Biochemistry, University of Leicester, Leicester, LE1 9HN, UK.
Eur Biophys J. 2009 Apr;38(4):395-405. doi: 10.1007/s00249-008-0383-z. Epub 2008 Nov 18.
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 Förster 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.
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。两个位置靠近的荧光团可能会导致福斯特共振能量转移（FRET），这种能量转移可以在单分子水平上被检测到，并且可以计算出能量转移效率。在大多数情况下，实验观察到的FRET效率分布呈现出显著的宽度，对应于0.07 - 0.2（范围为0 - 1）。在这里，我们提出了一种描述DNA/RNA双链体实验数据分析的通用方法。我们发现，对于一个15个碱基对的双链体，在螺旋相对两端连接了Cy3和Cy5荧光团，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，而染料间距离的变化起的作用较小。
