The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics.
The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics.
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DOI：
10.1038/ncomms8304
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发表时间：
2015-06-18
影响因子：
16.6
通讯作者：
Wuite GJ
中科院分区：
文献类型：
--
作者：
Biebricher AS;Heller I;Roijmans RF;Hoekstra TP;Peterman EJ;Wuite GJ
DNA嵌入剂被广泛用作荧光探针，用于显示体内和体外的DNA和DNA交易。众所周知，它们会扰乱DNA的结构和稳定性，进而影响蛋白质对DNA的加工。在这里，我们通过将单染料荧光显微镜与力谱相结合，并测量了单层和双层嵌入菁染料SYTOX橙、SYTOX绿、SYBR Gold、YO-PRO-1、YOYO-1和POPO-3对DNA的插层动力学，从而阐明了这种扰动。我们表明，它们的DNA结合亲和力主要由强烈依赖于张力的解离速率控制。这些速率可以通过改变DNA张力、嵌入物种和离子强度在七个数量级的范围内进行调节。我们表明，优化这些速率最大限度地减少了嵌入剂对链分离和酶活性的影响。这些新的见解为更好地使用嵌入剂作为DNA探针提供了处理，使其具有最小的扰动和最大的效率。DNA嵌入剂是一种被广泛用于显示DNA的荧光探针，它可以干扰DNA的结构和稳定性。在这里，作者展示了如何使用DNA张力、离子强度和染料种类来调节DNA结合亲和力，以及如何使用这一点来最大限度地减少DNA结构扰动。
DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro. It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy. DNA intercalators, a type of fluorescent probes widely used to visualize DNA, can perturb DNA structure and stability. Here, the authors show how DNA-binding affinity can be tuned using DNA tension, ionic strength and dye species, and how this can be used to minimize DNA structural perturbations.