Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules
Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules
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DOI：
10.1073/pnas.0401690101
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发表时间：
2004-06-15
影响因子：
11.1
通讯作者：
Weiss, S
中科院分区：
文献类型：
--
作者：
Kapanidis, AN;Lee, NK;Weiss, S
我们使用交替激光激发来实现荧光辅助分子分选（FAMS），并能够在单分子水平上同时分析生物分子结构和相互作用。这是通过用荧光团标记生物分子来进行的，荧光团用作Forster共振能量转移的供体-受体对，并且通过使用交替激光激发来直接激发单个扩散分子中存在的供体和受体。排放量减少到距离相关的比率E，以及距离无关的基于化学计量的比率S。基于每个物种的构象和缔合状态的E和S分类物种的直方图。S是敏感的化学计量和相对亮度的荧光团在单个分子，可观察到的低聚和局部环境的变化，分别。FAMS允许大分子-配体相互作用的平衡和动力学分析;这通过测量大肠杆菌分解代谢物激活蛋白与DNA相互作用的平衡和动力学解离常数来验证。FAMS是比率测量的通用平台，报告扩散或固定分子的结构、动力学、化学计量、环境和相互作用，从而实现详细的机理研究和超灵敏诊断。
We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of bionnolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor-acceptor pairs for Forster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics.