论文标题
DNA链位移的首次通过时间研究
First passage time study of DNA strand displacement
论文作者
论文摘要
DNA链位移,其中单链核酸侵入DNA双链体,在基因组过程和DNA工程应用中普遍存在。已批量研究了链位移的动力学。但是,基础链交换的动力学通过缓慢的双分子关联步骤掩盖。在这里,我们使用一种新型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道时间的完整分布。在4.4毫秒的框架时间内,14 nt位移域的第一个通道时间分布显示出几乎单调的衰减,几乎没有延迟。在我们测试的八个不同序列中,平均位移时间平均为35毫秒,并在13倍上变化。测得的位移动力学在互补入侵器之间以及相同基础序列的RNA和DNA侵入剂之间也有所不同,除了T $ \ rightarrow $ us替代。然而,位移时间在0.25 m至1m的范围内对单价盐浓度不敏感。使用一维随机步行模型,我们推断,单步位移时间在$ \ sim30μs$至$ \ sim \ sim300μs$的范围内。此处介绍的框架广泛适用于单分子水平研究的多步过程的动力学分析。
DNA strand displacement, where a single-stranded nucleic acid invades a DNA duplex, is pervasive in genomic processes and DNA engineering applications. The kinetics of strand displacement have been studied in bulk; however, the kinetics of the underlying strand exchange were obfuscated by a slow bimolecular association step. Here, we use a novel single-molecule Fluorescence Resonance Energy Transfer (smFRET) approach termed the "fission" assay to obtain the full distribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, the first passage time distribution for a 14-nt displacement domain exhibited a nearly monotonic decay with little delay. Among the eight different sequences we tested, the mean displacement time was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also varied between complementary invaders and between RNA and DNA invaders of the same base sequence except for T$\rightarrow$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 $\sim 30 μs$ to $\sim 300 μs$ 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.