DNA strand displacement, where a single-stranded nucleic acid invades a DNA duplex, is pervasive in genomic processes and DNA engineering applications. The kinetics of strand displacement have been studied in bulk; however, the kinetics of the underlying strand exchange were obfuscated by a slow bimolecular association step. Here, we use a novel single-molecule Fluorescence Resonance Energy Transfer (smFRET) approach termed the "fission" assay to obtain the full distribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, the first passage time distribution for a 14-nt displacement domain exhibited a nearly monotonic decay with little delay. Among the eight different sequences we tested, the mean displacement time was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also varied between complementary invaders and between RNA and DNA invaders of the same base sequence except for T→U substitution. However, displacement times were largely insensitive to the monovalent salt concentration in the range of 0.25 M to 1 M. Using a one-dimensional random walk model, we infer that the single-step displacement time is in the range of ~30 μs to ~300 μs depending on the base identity. The framework presented here is broadly applicable to the kinetic analysis of multistep processes investigated at the single-molecule level.

中文翻译：

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DNA链置换的首次通过时间研究

单链核酸侵入DNA双链体的DNA链置换普遍存在于基因组过程和DNA工程应用中。大量研究了股线位移的动力学。然而，潜在的链交换的动力学被缓慢的双分子缔合步骤所混淆。在这里，我们使用一种新颖的单分子荧光共振能量转移（smFRET）方法，称为“裂变”测定法，以获得单分子链位移的第一个通过时间的完整分布。在4.4 ms的帧时间上，14 nt位移域的第一次通过时间分布表现出几乎单调的衰减，几乎没有延迟。在我们测试的八种不同序列中，平均置换时间平均为35 ms，变化幅度最大为13。除了T→U取代，在相同的碱基序列的互补入侵者之间以及RNA和DNA入侵者之间，测得的置换动力学也有所不同。但是，位移时间对0.25 M至1 M范围内的单价盐浓度非常不敏感。使用一维随机游走模型，我们推断单步位移时间在〜30μs至〜范围内。 300μs，取决于碱基识别。本文介绍的框架可广泛应用于在单分子水平上研究的多步过程的动力学分析。使用一维随机游走模型，我们推断单步位移时间在约30μs到〜300μs的范围内，具体取决于碱基身份。本文介绍的框架可广泛应用于在单分子水平上研究的多步过程的动力学分析。使用一维随机游走模型，我们推断单步位移时间在约30μs到〜300μs的范围内，具体取决于碱基身份。本文介绍的框架可广泛应用于在单分子水平上研究的多步过程的动力学分析。