Probing the structure and dynamics of a DNA hairpin by ultrafast quenching and fluorescence depolarization
Probing the structure and dynamics of a DNA hairpin by ultrafast quenching and fluorescence depolarization
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DOI：
10.1016/s0006-3495(01)75768-2
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发表时间：
2001-08-01
影响因子：
3.4
通讯作者：
van Amerongen, H
中科院分区：
文献类型：
--
作者：
Larsen, OFA;van Stokkum, IHM;van Amerongen, H
已经研究了DNA发夹，其中单个腺嘌呤被其荧光类似物2-氨基嘌呤（2AP）取代。利用皮秒时间分辨率监测了偏振发射光谱随时间的演化。每个寡核苷酸的四个各向同性衰变组分表明至少四种构象的共存。其中三种的荧光被显著淬灭，这可以通过从2AP到鸟嘌呤的空穴转移来解释。类似于8-ps的成分归因于直接空穴传输，类似于50-ps和类似于500-ps的成分归因于结构重组，先于空穴传输。在室温下，一个馏分保持在10 ns的时间尺度上未淬灭，在较高的温度下，在那里的灵活性增加。除了由于碱基堆积导致的猝灭之外，还需要第二猝灭过程来描述数据。发现了链内和链间空穴转移的证据。在双链区域中相邻碱基之间堆叠的提取概率估计在室温下为75%，在80 degreesC下为25%，表明DNA的结构紊乱。荧光去极化揭示了DNA的局部动力学和整个寡核苷酸的整体动力学。在升高温度时，发夹的C-N末端似乎首先熔化;发夹的其余部分在平均熔化温度以上变性。
DNA hairpins have been investigated in which individual adenines were replaced by their fluorescent analog 2-aminopurine (2AP). The temperature dependence of the time evolution of polarized emission spectra was monitored with picosecond time resolution. Four isotropic decay components for each oligonucleotide indicated the coexistence of at least four conformations. The fluorescence for three of these was significantly quenched, which is explained by hole transfer from 2AP to guanine(s). An similar to8-ps component is ascribed to direct hole transfer, the similar to 50-ps and similar to 500-ps components are ascribed to structural reorganization, preceding hole transfer. At room temperature, a fraction remains unquenched on a 10-ns timescale, in contrast to higher temperatures, where the flexibility increases. Besides quenching due to base stacking, a second quenching process was needed to describe the data. Evidence for both intrastrand and interstrand hole transfer was found. The extracted probability for stacking between neighboring bases in double-stranded regions was estimated to be similar to 75% at room temperature and similar to 25% at 80 degreesC, demonstrating structural disorder of the DNA. Fluorescence depolarization revealed both local dynamics of the DNA and overall dynamics of the entire oligonucleotide. Upon raising the temperature, the C-N terminus of the hairpin appears to melt first; the rest of the hairpin denatures above the average melting temperature.