Probing DNA Dynamics: Stacking-Induced Fluorescence Increase (SIFI) versus FRET
Probing DNA Dynamics: Stacking-Induced Fluorescence Increase (SIFI) versus FRET
复制标题
探测 DNA 动力学：堆积诱导荧光增加 (SIFI) 与 FRET
DOI：
10.1002/cptc.202000069
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发表时间：
2020
期刊：
影响因子：
3.7
通讯作者：
Morten M
中科院分区：
文献类型：
--
作者：
Morten M
堆叠诱导荧光增加（SIFI）是最近引入的一种仅使用单个荧光标记来探测DNA结构和动力学的方法。在单分子水平上，使用SIFI（以Cy3为标签）或FRET（以Cy3为供体，Cy5为受体）可以恢复相同的DNA发夹动力学。我们还使用在存在和不存在分子拥挤剂（PEG）的情况下不能进行SIFI的供体Cy3B来测量FRET。虽然拥挤增加发夹杂交的程度与作为供体的Cy3或Cy3B相同，但绝对速率受供体染料选择的影响。这项工作表明，SIFI可以用于测量单分子动力学，这在某些情况下可能比FRET更具优势。它还说明了局部染料相互作用如何影响生物分子动力学，这在设计实验时应该考虑。
Stacking‐induced fluorescence increase (SIFI) was introduced recently as a method to probe DNA structure and dynamics using only a single fluorescent label. Here we show that the same DNA hairpin dynamics can be recovered, at the single‐molecule level, using either SIFI (with Cy3 as the label) or FRET (with Cy3 as donor and Cy5 as acceptor). We also measured FRET using a donor that cannot undergo SIFI, Cy3B, in the presence and absence of a molecular crowding agent (PEG). Although crowding increases hairpin hybridisation to the same extent with either Cy3 or Cy3B as the donor, the absolute rates are affected by the choice of donor dye. This work shows that SIFI can be used to measure single‐molecule dynamics, which could offer advantages over FRET in some cases. It also illustrates how local dye interactions can influence biomolecular dynamics, which should be considered when designing experiments.