Single-molecule structural and kinetic studies across sequence space

Science, vol. 385, pp. 898–904

Abstract

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.

Authors 6

1. Ivo Severins Aachen Biological and Soft Matter Physics Huygens-Kamerlingh Onnes Laboratory

   Leiden University · Delft University of Technology

   Affiliation as printed

   Biological and Soft Matter Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, Netherlands

   Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands

2. Carolien Bastiaanssen

   Delft University of Technology

   Affiliation as printed

   Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands

3. Sung Hyun Kim

   Ewha Womans University · Delft University of Technology

   Affiliation as printed

   Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands

   Department of Physics, Ewha Womans University, Seoul 03760, Republic of Korea

4. Roy B. Simons

   Delft University of Technology

   Affiliation as printed

   Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands

5. John van Noort corresponding Aachen Biological and Soft Matter Physics Huygens-Kamerlingh Onnes Laboratory

   Leiden University

   Affiliation as printed

   Biological and Soft Matter Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, Netherlands

6. Chirlmin Joo corresponding

   Ewha Womans University · Delft University of Technology

   Affiliation as printed

   Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, the Netherlands

   Department of Physics, Ewha Womans University, Seoul 03760, Republic of Korea