Department of Chemistry , University of Utah , 315 South 1400 East , Salt Lake City , Utah 84112-0850 , United States.
J Phys Chem B. 2019 Dec 19;123(50):10746-10756. doi: 10.1021/acs.jpcb.9b09736. Epub 2019 Dec 4.
Single-molecule fluorescence imaging is a powerful method to measure reversible reaction kinetics, allowing one to monitor the bound state of individual probe molecules with fluorescently labeled targets. In the case of DNA hybridization, previous studies have shown that the presence of a fluorescent label on a target strand can exhibit significant influence on the stability of a DNA duplex that is formed. In this work, we have developed a super-resolution imaging method to measure the hybridization kinetics of unlabeled target DNA that compete with a fluorescently labeled tracer DNA strand to hybridize with an unlabeled probe DNA immobilized at a surface. The hybridization of an unlabeled DNA target cannot be detected directly, but its presence blocks the immobilized probe DNA, influencing the measured time intervals between labeled DNA hybridization events. We derive a model for competitive hybridization kinetics to extract the association and dissociation rate constants of the unlabeled species from the distribution of time intervals between hybridization events of the labeled tracer DNA at individual localized DNA probe sites. We use this methodology to determine the hybridization kinetics of a model 11-mer unlabeled target DNA strand and then determine how five different fluorescent labels attached to the same target DNA strand impact the hybridization kinetics. Compared to the unlabeled target, these labels can slow the association and dissociation rates by as much as a factor of 5. The super-resolution time-interval methodology provides a unique approach to determining fundamental (label-free) rates of DNA hybridization, revealing the significant influence of fluorescent labels on these kinetics. This measurement concept can be extended to studies of other reversible reaction systems, where kinetics of unlabeled species can be determined from their influence on the reaction of a labeled species with localized probe molecules on a surface.
单分子荧光成像技术是一种强大的方法来测量可逆反应动力学，允许一个监测与荧光标记的目标个别探针分子的结合状态。在 DNA 杂交的情况下，以前的研究表明，一个目标链上的荧光标记的存在可以表现出显著的影响，对一个 DNA 双链体的稳定性，形成。在这项工作中，我们已经开发了一种超分辨率成像方法来测量杂交动力学的未标记的目标 DNA 与竞争一个荧光标记示踪 DNA 链杂交与一个未标记的探针 DNA 固定在表面上。杂交的未标记的 DNA 目标不能直接检测到，但它的存在阻止了固定的探针 DNA，影响了测量的时间间隔之间的标记 DNA 杂交事件。我们推导出一个模型的竞争杂交动力学来提取关联和离解速率常数的未标记的物种从分布的时间间隔之间的杂交事件的标记示踪 DNA 在个别本地化 DNA 探针网站。我们使用这种方法来确定杂交动力学的一个模型 11-mer 未标记的目标 DNA 链，然后确定如何五个不同的荧光标记附着到相同的目标 DNA 链影响杂交动力学。与未标记的目标，这些标签可以减缓关联和离解速率高达 5 倍。超分辨率时间间隔的方法提供了一种独特的方法来确定基本的（无标记）的 DNA 杂交速率，揭示了荧光标记对这些动力学的显著影响。这种测量的概念可以扩展到其他可逆反应体系的研究，其中未标记的物种的动力学可以从他们的影响与一个标记的物种与局部探针分子在表面上的反应来确定。
