Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Forster distance dependence and subpopulations
Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Forster distance dependence and subpopulations
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DOI：
10.1073/pnas.96.7.3670
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发表时间：
1999-03-30
影响因子：
11.1
通讯作者：
Schultz, PG
中科院分区：
文献类型：
--
作者：
Deniz, AA;Dahan, M;Schultz, PG
从单个扩散供体-受体标记的大分子的光子爆发被用来测量分子内距离，并确定在异质系综中自由扩散的大分子的亚群。通过使用DNA作为刚性间隔子，一系列具有不同分子内供体-受体间距的构建体被用于测量作为距离的函数的荧光共振能量转移（FRET）效率的平均值和分布宽度。平均单对FRET效率定性地遵循福斯特理论预测的距离依赖性。FRET效率分布的宽度可能的贡献进行了讨论，并建议在生物聚合物构象动力学研究的潜在应用。测量单个分子的分子内（和分子间）距离的能力意味着区分和监测混合物中具有不同距离或构象状态的分子亚群的能力。这通过在限制性内切酶切割反应之前和之后监测底物和产物亚群来证明。单分子分辨率的距离测量也应该有助于研究复杂的反应，如生物聚合物折叠。为此，通过使用单对FRET检查DNA发夹的变性。
Photon bursts from single diffusing donor-acceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of fluorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Forster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single pair FRET.