Single-molecule parallel analysis for rapid exploration of sequence space
C.K.J.M.L. Bastiaanssen (TU Delft - BN/Chirlmin Joo Lab, Kavli institute of nanoscience Delft)
I.W.H. Severins (TU Delft - BN/Chirlmin Joo Lab, Kavli institute of nanoscience Delft, TU Delft - BN/Bionanoscience, Ewha Womans University, Universiteit Leiden, TU Delft - Sports & Games)
John van Noort (Universiteit Leiden)
C. Joo (TU Delft - BN/Bionanoscience, TU Delft - BN/Chirlmin Joo Lab, Ewha Womans University, Kavli institute of nanoscience Delft)
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Abstract
Single-molecule fluorescence techniques have been successfully applied to uncover the structure, dynamics and interactions of DNA, RNA and proteins at the molecular scale. While the structure and function of these biomolecules are imposed by their sequences, single-molecule studies have been limited to a small number of sequences due to constraints in time and cost. To gain a comprehensive understanding on how sequence influences these essential biomolecules and the processes in which they act, a vast number of sequences have to be probed, requiring a high-throughput parallel approach. To address this need, we developed SPARXS: single-molecule parallel analysis for rapid exploration of sequence space. This platform enables simultaneous profiling of millions of molecules, covering thousands of distinct sequences, at the single-molecule level by coupling single-molecule fluorescence microscopy with next-generation high-throughput sequencing. Here we describe how to implement SPARXS and give examples from our study into the effect of sequence on Holliday junction kinetics. We provide a detailed description of sample and library design, single-molecule measurement, sequencing, coupling of sequencing and single-molecule fluorescence data, and data analysis. The protocol requires experience with single-molecule fluorescence microscopy and a basic command of Python to use our Papylio package for SPARXS data analysis. Familiarity with the underlying principles of Illumina sequencing is also beneficial. The entire process takes ~1–2 weeks and provides a detailed quantitative picture of the effect of sequence on the studied process.