At the core of molecular biology lies the intricate interplay between sequence, structure, and function. Single-molecule techniques provide in-depth dynamic insights into structure and function, but laborious assays impede functional screening of large sequence libraries. We introduce high-throughput Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS), integrating single-molecule fluorescence with next-generation sequencing. We applied SPARXS to study the sequence-dependent kinetics of the Holliday junction, a critical intermediate in homologous recombination. By examining the dynamics of millions of Holliday junctions, covering thousands of distinct sequences, we demonstrated the ability of SPARXS to uncover sequence patterns, evaluate sequence motifs, and construct thermodynamic models. SPARXS emerges as a versatile tool for untangling the mechanisms that underlie sequence-specific processes at the molecular scale.

中文翻译：

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跨序列空间的单分子结构和动力学研究


分子生物学的核心在于序列、结构和功能之间复杂的相互作用。单分子技术提供了对结构和功能的深入动态洞察，但费力的测定阻碍了大型序列库的功能筛选。我们引入了用于快速探索序列空间的高通量单分子并行分析 (SPARXS)，将单分子荧光与下一代测序相结合。我们应用 SPARXS 来研究霍利迪连接体（同源重组中的关键中间体）的序列依赖性动力学。通过检查数百万个霍利迪连接点的动态，涵盖数千个不同的序列，我们证明了 SPARXS 揭示序列模式、评估序列基序和构建热力学模型的能力。 SPARXS 成为一种多功能工具，用于解开分子尺度上序列特异性过程背后的机制。