Massively parallel analysis of single-molecule dynamics on next-generation sequencing chips

• 成果类型: Article
• 署名作者: Rivera, J. Aguirre; Mao, G.; Sabantsev, A.; Panfilov, M.; Hou, Q.; Lindell, M.; Chanez, C.; Ritort, F.; Jinek, M.; Deindl, S.
• 署名单位: Uppsala University; Uppsala University; University of Zurich; University of Barcelona; University of Barcelona
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-13625
• DOI: 10.1126/science.adn5371
• 发表日期: 2024-08-23
• 页码: 892-898
• 关键词: rna-protein interactions; conformational dynamics; magnetic tweezers; target cleavage; energy-transfer; dna cleavage; fret; crispr-cas9; reveals; transcription

摘要

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.