Single-molecule structural and kinetic studies across sequence space

• 成果类型: Article
• 署名作者: Severins, Ivo; Bastiaanssen, Carolien; Kim, Sung Hyun; Simons, Roy B.; van Noort, John; Joo, Chirlmin
• 署名单位: Delft University of Technology; Leiden University - Excl LUMC; Leiden University; Ewha Womans University
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-13626
• DOI: 10.1126/science.adn5968
• 发表日期: 2024-08-23
• 页码: 898-904
• 关键词: double holliday junctions; branch migration; dna curtains; DYNAMICS; identification

摘要

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.