Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules.
Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules.
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DOI：
10.1371/journal.pone.0175766
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Michalet X
中科院分区：
文献类型：
--
作者：
Ingargiola A;Lerner E;Chung S;Panzeri F;Gulinatti A;Rech I;Ghioni M;Weiss S;Michalet X
我们描述了一个8点共聚焦设置高通量smFRET测定和说明其性能与两个特征实验。首先，对一系列自由扩散的双标记dsDNA样品的测量使我们能够证明，在多个平行点上获得的数据可以得到适当的校正，并导致测量的样品特征与标准单点设置获得的特征一致。然后，我们利用平行获取提供的更高通量来解决关于细菌RNA转录初始步骤动力学的突出问题。我们对细菌RNA聚合酶启动子逃逸的实时动力学分析证实了通过更间接的途径获得的结果，为转录的初始步骤提供了更多的信息。最后，我们讨论了我们的多点设置的优点，同时指出了当前单激光激发设计的潜在局限性，以及分析挑战和解决方案。
We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.