Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profiled the sequence dependence of DNA hairpin properties and Cas9-induced target DNA unwinding-rewinding dynamics. The ability to explore a large sequence space for Cas9 allowed us to identify a number of target sequences with unexpected behaviors. We envision that MUSCLE will enable the mechanistic exploration of many fundamental biological processes.

中文翻译：

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下一代测序芯片上单分子动力学的大规模并行分析


单分子技术非常适合表征复杂的动力学，但通常仅限于研究少量不同的样品。然而，通常需要探索大的序列或化学空间才能全面了解复杂的生物过程。在这里，我们描述了文库规模的多重单分子表征（MUSCLE），这是一种将单分子荧光显微镜与下一代测序相结合的方法，能够对复杂动力学进行高度多重观察。我们全面分析了 DNA 发夹特性的序列依赖性和 Cas9 诱导的靶 DNA 解旋-重绕动力学。探索 Cas9 的大序列空间的能力使我们能够识别出许多具有意外行为的目标序列。我们设想 MUSCLE 将使许多基本生物过程的机械探索成为可能。