Sabanayagam Chandran R, Eid John S, Meller Amit
Rowland Institute at Harvard, Harvard University, Cambridge, Massachusetts 02142, USA.
J Chem Phys. 2005 Feb 8;122(6):061103. doi: 10.1063/1.1854120.
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 approximately 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.
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，由于涉及染料多种去激发途径的复杂性，很少能获得染料间的绝对距离信息。为了规避这一问题，我们直接探测单个荧光团量子产率的相对变化。以B-DNA作为支架，将供体（Cy3或TMR）置于福斯特半径范围内距受体（Cy5）精确距离处。我们发现，Cy3量子产率的变化比TMR的大约大5倍。通过考虑受体/供体量子产率比中分子间的变异性，对表观荧光共振能量转移效率进行缩放以得到理论值。我们与预测沿B-DNA距离的物理模型取得了很好的一致性。
