Deniz A A, Dahan M, Grunwell J R, Ha T, Faulhaber A E, Chemla D S, Weiss S, Schultz P G
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3670-5. doi: 10.1073/pnas.96.7.3670.
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 Förster 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.
来自单个扩散的供体 - 受体标记大分子的光子爆发被用于测量分子内距离，并识别异质集合中自由扩散大分子的亚群。通过使用DNA作为刚性间隔物，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荧光共振能量转移（FRET）效率的平均值和分布宽度作为距离的函数。平均单对FRET效率定性地遵循Förster理论预测的距离依赖性。讨论了对FRET效率分布宽度的可能贡献，并提出了在生物聚合物构象动力学研究中的潜在应用。测量单个分子的分子内（和分子间）距离的能力意味着能够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。这通过监测限制性内切酶切割反应前后的底物和产物亚群得到了证明。单分子分辨率下的距离测量也应有助于研究诸如生物聚合物折叠等复杂反应。为此，通过使用单对FRET研究了DNA发夹的变性。
