Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands.
Biological and Soft Matter Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, Netherlands.
Science. 2024 Aug 23;385(6711):898-904. doi: 10.1126/science.adn5968. Epub 2024 Aug 22.
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.
在分子生物学的核心，序列、结构和功能之间存在着错综复杂的相互作用。单分子技术为结构和功能提供了深入的动态见解，但繁琐的检测阻碍了对大型序列文库的功能筛选。我们引入了高通量单分子平行分析快速探索序列空间（SPARXS），将单分子荧光与下一代测序相结合。我们应用 SPARXS 来研究霍利迪交叉（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交叉的动态，涵盖了数千个不同的序列，我们展示了 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 成为了一种通用工具，可以理清分子尺度上序列特异性过程背后的机制。
