Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer
Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer
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DOI：
10.1063/1.1854120
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发表时间：
2005-02-08
影响因子：
4.4
通讯作者：
Meller, A
中科院分区：
文献类型：
--
作者：
Sabanayagam, CR;Eid, JS;Meller, A
单分子荧光共振能量转移已被广泛用于测量各种生物分子系统中的距离变化和动力学。然而，由于涉及染料的多个去激发途径的复杂性，染料间的绝对距离信息很少被恢复。为了避免这一点，我们直接探测单个荧光团的量子产率的相对变化。使用B-DNA作为支架，将供体（Cy 3或TMR）定位在福斯特半径内与受体（Cy 5）的精确距离处。我们发现Cy 3量子产率的变化是TMR的5倍。通过考虑受体/供体量子产率比的分子-分子可变性，表观荧光共振能量转移效率被缩放以产生理论值。我们与预测沿着B-DNA距离的物理模型获得了非常好的一致性。(C)单分子荧光共振能量转移已被广泛用于测量各种生物分子系统中的距离变化和动力学。然而，由于涉及染料的多个去激发途径的复杂性，染料间的绝对距离信息很少被恢复。为了避免这一点，我们直接探测单个荧光团的量子产率的相对变化。使用B-DNA作为支架，将供体（Cy 3或TMR）定位在福斯特半径内与受体（Cy 5）的精确距离处。我们发现Cy 3量子产率的变化比TMR大5倍。通过考虑受体/供体量子产率比的分子-分子可变性，表观荧光共振能量转移效率被缩放以产生理论值。我们与预测沿着B-DNA距离的物理模型获得了非常好的一致性。
Single molecule fluorescence resonance energy transfer has been extensively used to measure distance changes and kinetics in various biomolecular systems. However, due to complications involving multiple de-excitation pathways of the dyes, the absolute inter-dye distance information has seldom been recovered. To circumvent this we directly probe the relative variations in the quantum yield of individual fluorophores. B-DNA was used as a scaffold to position the donor (Cy3 or TMR) at precise distances from the acceptor (Cy5) within the Forster radius. We found that the variation in the Cy3 quantum yield is,5 times larger than that of TMR. By taking into account the molecule-to-molecule variability in the acceptor/donor quantum yield ratio, the apparent fluorescence resonance energy transfer efficiencies were scaled to yield the theoretical values. We obtained very good agreement with a physical model that predicts distances along B-DNA. (C) 2005 American Institute of Physics.Single molecule fluorescence resonance energy transfer has been extensively used to measure distance changes and kinetics in various biomolecular systems. However, due to complications involving multiple de-excitation pathways of the dyes, the absolute inter-dye distance information has seldom been recovered. To circumvent this we directly probe the relative variations in the quantum yield of individual fluorophores. B-DNA was used as a scaffold to position the donor (Cy3 or TMR) at precise distances from the acceptor (Cy5) within the Forster radius. We found that the variation in the Cy3 quantum yield is similar to5 times larger than that of TMR. By taking into account the molecule-to-molecule variability in the acceptor/donor quantum yield ratio, the apparent fluorescence resonance energy transfer efficiencies were scaled to yield the theoretical values. We obtained very good agreement with a physical model that predicts distances along B-DNA.