Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy
Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy
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DOI：
10.1073/pnas.95.4.1556
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发表时间：
1998-02-17
影响因子：
11.1
通讯作者：
Seidel, CAM
中科院分区：
文献类型：
--
作者：
Eggeling, C;Fries, JR;Seidel, CAM
一种近期开发的实时光谱技术——爆发积分荧光寿命（BIFL），被证明非常适合监测单个分子在共焦落射照明装置的微观开放测量体积（约10飞升）中扩散时的单个分子构象动力学。在一种用环境敏感荧光染料四甲基罗丹明（TMR）标记的20聚体DNA双链寡核苷酸链的高度稀释水溶液中，记录表明单个分子踪迹的荧光爆发，并进一步进行选择性爆发分析。二维BIFL数据能够识别和检测不同的时间分辨构象状态。对荧光寿命和强度随时间的波动进行了互补的自相关分析。一致的结果有力地支持了所假设的TMR - DNA双链构象动力学的三态模型，即TMR具有极性、非极性和猝灭环境。
A recently developed, real-time spectroscopic technique, burst-integrated fluorescence lifetime (BIFL), is shown to be well suited for monitoring the individual molecular conformational dynamics of a single molecule diffusing through the microscopic, open measurement volume (approximate to 10 fl) of a confocal epi-illuminated set-up, In a highly diluted aqueous solution of 20-mer oligonucleotide strand of DNA duplex labeled with the environment-sensitive fluorescent dye tetramethylrhodamine (TMR), fluorescence bursts indicating traces of individual molecules are registered and further subjected to selective burst analysis, The two-dimensional BIEL data allow the identification and detection of different temporally resolved conformational states, A complementary autocorrelation analysis was performed on the time-dependent fluctuations in fluorescence lifetime and intensity, The consistent results strongly support the hypothesized three-state model of the conformational dynamics of the TMR-DNA duplex with a polar, a nonpolar, and a quenching environment of TMR.