Professor Sebastian Deindl, Department of Cell and Molecular Biology, Uppsala University

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

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. We have developed a method, MuSCLe, that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profile the sequence dependence of DNA hairpin dynamics and Cas9-induced target DNA unwinding/rewinding dynamics. For Cas9, the ability to explore a large sequence space allowed us to identify a number of target sequences with unexpected behaviors that cannot be explained by canonical base pairing. We envision that MuSCLe will enable the mechanistic exploration of many fundamental biological processes.