Probing DNA Dynamics: Stacking‐Induced Fluorescence Increase (SIFI) versus FRET,ChemPhotoChem

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Probing DNA Dynamics: Stacking‐Induced Fluorescence Increase (SIFI) versus FRET
ChemPhotoChem ( IF 3.0 ) Pub Date : 2020-04-17 , DOI: 10.1002/cptc.202000069
Michael J. Morten ₁ , I. Emilie Steinmark ₁ , Steven W. Magennis ₁

Affiliation

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.

中文翻译：

探测DNA动力学：堆积诱导荧光增加（SIFI）与FRET

最近引入了堆积诱导的荧光增加（SIFI）作为仅使用单个荧光标记物即可探测DNA结构和动力学的方法。在这里我们表明，使用SIFI（以Cy3为标记）或FRET（以Cy3为供体，以Cy5为受体），可以在单分子水平上恢复相同的DNA发夹动力学。我们还使用存在和不存在分子拥挤剂（PEG）的情况下，使用不能经历SIFI的供体Cy3B来测量FRET。尽管拥挤增加了与供体使用Cy3或Cy3B进行发夹杂交的程度，但绝对速率受供体染料的选择影响。这项工作表明，SIFI可用于测量单分子动力学，在某些情况下，它可以提供优于FRET的优势。它还说明了局部染料相互作用如何影响生物分子动力学，

更新日期：2020-04-17

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