Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids.
Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids.
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DOI：
10.1002/bip.10146
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Dagmar Klostermeier;David P. Millar
中科院分区：
文献类型：
--
作者：
Dagmar Klostermeier;David P. Millar
核酸在DNA复制、转录、同源重组、mRNA翻译和核酶催化等过程中的生物学功能与其三维结构密切相关，并与蛋白质、金属离子和其他配体引起的构象变化密切相关。荧光光谱是一种在广泛的溶液条件下探测生物大分子的结构和构象动力学的强大技术。荧光共振能量转移(FRET)提供了从10A到100A的远程信息，这一范围对于探测核酸的全球结构很有用。虽然FRET的稳态测量提供了施主和受体之间的平均距离，但从时间分辨FRET(TrFRET)实验中对施主纳秒发射衰减的分析可以获得更多的信息。根据供体-受体距离分布分析衰变可以解析多相混合物中的不同构象，提供关于每个物种及其平衡种群的全局结构和灵活性的信息。在这篇综述中，我们概述了trFRET的原理以及将荧光探针掺入DNA和RNA中的方法。举例说明了trFRET作为定义整体结构、识别构象异质性和灵活性、研究三级结构形成的能量学和探索核酸结构重排的工具的多功能性。
The biological functions of nucleic acids in processes of DNA replication, transcription, homologous recombination, mRNA translation, and ribozyme catalysis are intimately linked to their three-dimensional structures and to conformational changes induced by proteins, metal ions and other ligands. Fluorescence spectroscopy is a powerful technique for probing the structure and conformational dynamics of biological macromolecules under a wide range of solution conditions. Fluorescence resonance energy transfer (FRET) provides long-range distance information from 10 to 100 A, a range that is useful for probing the global structure of nucleic acids. While steady-state measurements of FRET provide the average distance between donor and acceptor, much more information is available from the analysis of the nanosecond emission decay of the donor in time-resolved FRET (trFRET) experiments. Analysis of the decay in terms of donor-acceptor distance distributions can resolve different conformers in a heterogeneous mixture, providing information on the global structure and flexibility of each species as well as their equilibrium populations. In this review, we outline the principles of trFRET and the methods used to incorporate fluorescent probes into DNA and RNA. Examples of specific applications are presented to illustrate the versatility of trFRET as a tool to define global structures, to identify conformational heterogeneity and flexibility, to investigate the energetics of tertiary structure formation and to probe structural rearrangements of nucleic acids.