Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation
Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation
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DOI：
10.1529/biophysj.104.054114
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发表时间：
2005-04-01
影响因子：
3.4
通讯作者：
Weiss, S
中科院分区：
文献类型：
--
作者：
Lee, NK;Kapanidis, AN;Weiss, S
供体（D）和受体（A）之间在单分子水平上的荧光共振能量转移（FRET）目前提供关于距离的定性信息和关于距离变化的动力学的定量信息。在这里，我们使用的分选能力的共聚焦显微镜配备了交替激光激发（ALEX），以测量准确的FRET效率和距离单分子，使用校正，占串扰的污染FRET诱导的信号，并在检测效率和量子产率的差异的探针。ALEX产生精确的FRET独立于仪器因素，如激发强度或检测器对齐。使用DNA片段，我们表明，ALEX为基础的距离同意以及从一个圆柱形的DNA模型的预测，ALEX为基础的距离更适合理论比在合奏水平上获得的距离。转录复合物内的距离测量同意与ensemble-FRET测量，并与ensemble-FRET和X-射线晶体学的基础上的结构模型。ALEX可以有益于生物分子的结构分析，特别是当这些分子由于异质性或瞬时性质而无法用常规结构方法进行分析时。
Fluorescence resonance energy transfer (FRET) between a donor (D) and an acceptor (A) at the single-molecule level currently provides qualitative information about distance, and quantitative information about kinetics of distance changes. Here, we used the sorting ability of confocal microscopy equipped with alternating-laser excitation (ALEX) to measure accurate FRET efficiencies and distances from single molecules, using corrections that account for cross-talk terms that contaminate the FRET-induced signal, and for differences in the detection efficiency and quantum yield of the probes. ALEX yields accurate FRET independent of instrumental factors, such as excitation intensity or detector alignment. Using DNA fragments, we showed that ALEX-based distances agree well with predictions from a cylindrical model of DNA; ALEX-based distances fit better to theory than distances obtained at the ensemble level. Distance measurements within transcription complexes agreed well with ensemble-FRET measurements, and with structural models based on ensemble-FRET and x-ray crystallography. ALEX can benefit structural analysis of biomolecules, especially when such molecules are inaccessible to conventional structural methods due to heterogeneity or transient nature.