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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity
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Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity

机译：蛋白诱导的荧光增强作为具有短距离灵敏度的单分子检测

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摘要

Single-molecule FRET has been widely used for monitoring protein–nucleic acids interactions. Direct visualization of the interactions, however, often requires a site-specific labeling of the protein, which can be circuitous and inefficient. In addition, FRET is insensitive to distance changes in the 0–3-nm range. Here, we report a systematic calibration of a single molecule fluorescence assay termed protein induced fluorescence enhancement. This method circumvents protein labeling and displays a marked distance dependence below the 4-nm distance range. The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a protein. Our data reveals that the method can resolve as small as a single base pair distance at the extreme vicinity of the fluorophore, where the enhancement is maximized. We demonstrate the general applicability and distance sensitivity using (a) a finely spaced DNA ladder carrying a restriction site for BamHI, (b) RNA translocation by DExH enzyme RIG‐I, and (c) filament dynamics of RecA on single-stranded DNA. The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.
机译：单分子FRET已广泛用于监测蛋白质-核酸相互作用。然而，相互作用的直接可视化通常需要蛋白质的位点特异性标记，这可能是circuit回的且效率低下。此外，FRET对0–3 nm范围内的距离变化不敏感。在这里，我们报告称为蛋白质诱导的荧光增强的单分子荧光测定的系统校准。此方法绕过蛋白质标记，并在4 nm距离范围以下显示明显的距离依赖性。荧光的增强基于光物理现象，由此荧光团的强度随着蛋白质的近端结合而增加。我们的数据表明，该方法在最大程度增强荧光的荧光基团的最邻近处可以分辨出单个碱基对的距离。我们使用（a）带有BamHI限制性酶切位点的细间距DNA阶梯，（b）DExH酶RIG-I进行RNA移位以及（c）RecA在单链DNA上的细丝动力学来证明其普遍适用性和距离敏感性。高时空分辨率数据和对短距离的敏感性以及绕过蛋白质标记的能力使该测定成为FRET的有效替代或补充。

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