Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy
Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy
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DOI：
10.1016/s0006-3495(04)74308-8
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发表时间：
2004-04-01
影响因子：
3.4
通讯作者：
Ha, TJ
中科院分区：
文献类型：
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作者：
Murphy, MC;Rasnik, I;Ha, TJ
单链DNA(Single-stranded DNA，ssDNA)是DNA代谢过程中的重要中间体，与大量蛋白质相互作用。由于其灵活性，单链DNA在溶液中的构象只能用统计方法来描述，如柔性连接链模型或蠕虫链模型。然而，可用于定量评估此类模型的数据有限，尤其是用于描述短单链DNA和RNA的灵活性。为了解决这个问题，我们在广泛的盐浓度和链长(10小于或等于N小于或等于70个核苷酸)范围内对一系列寡聚脱氧胸腺苷酸酯(DT)(N)进行了FRET研究，这为测试理论模型提供了系统的限制。在机械研究中，可用的单链DNA构象是在进行测量所需的时间内平均出来的，与此不同的是，荧光寿命在这里可能作为一个内部时钟，根据单链DNA构象波动的速度影响荧光信号。假设在给体的荧光寿命内发生了单链DNA构象的有限弛豫，我们的数据与蠕虫链模型之间可以获得相当好的一致性。由此估计的持续长度在2M的氯化钠中为1.5 nm，在25 mM的氯化钠中为3 nm。
Single-stranded DNA (ssDNA) is an essential intermediate in various DNA metabolic processes and interacts with a large number of proteins. Due to its flexibility, the conformations of ssDNA in solution can only be described using statistical approaches, such as flexibly jointed or worm-like chain models. However, there is limited data available to assess such models quantitatively, especially for describing the flexibility of short ssDNA and RNA. To address this issue, we performed FRET studies of a series of oligodeoxythymidylates, (dT)(N), over a wide range of salt concentrations and chain lengths (10 less than or equal to N less than or equal to 70 nucleotides), which provide systematic constraints for testing theoretical models. Unlike in mechanical studies where available ssDNA conformations are averaged out during the time it takes to perform measurements, fluorescence lifetimes may act here as an internal clock that influences fluorescence signals depending on how fast the ssDNA conformations fluctuate. A reasonably good agreement could be obtained between our data and the worm-like chain model provided that limited relaxations of the ssDNA conformations occur within the fluorescence lifetime of the donor. The persistence length thus estimated ranges from 1.5 nm in 2 M NaCl to 3 nm in 25 mM NaCl.