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Hydronium and hydroxide ions are present both in pure water and in all aqueous solutions, and their concentrations are inversely proportional as determined by the ion product of water (Kw). The concentrations of these ions in a solution are often critical determinants of the solution’s properties and the chemical behaviors of its other solutes. Two different solutions can differ in their hydronium or hydroxide ion concentrations by a million, billion, or even trillion times. A common means of...
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Multiplexed single-molecule characterization at the library scale.

M Panfilov1, G Mao1, J Guo1

  • 1Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

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|June 4, 2025
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Multiplexed Single-Molecule Characterization at the Library Scale (MUSCLE) enables high-throughput analysis of millions of molecules. This method combines single-molecule fluorescence microscopy and next-generation sequencing for comprehensive dynamic behavior characterization.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Biochemistry

Background:

  • Single-molecule techniques are crucial for understanding macromolecular dynamics in biological processes.
  • Current methods are limited by time and cost, restricting the analysis of diverse samples.
  • Investigating broad sequence or chemical spaces is essential for a comprehensive understanding of biological phenomena.

Purpose of the Study:

  • To develop a high-throughput method for characterizing the dynamic behavior of large libraries of molecules.
  • To overcome the limitations of existing single-molecule techniques in terms of sample number and scale.
  • To enable the study of complex dynamics across thousands of distinct sequences or barcoded entities.

Main Methods:

  • Multiplexed Single-Molecule Characterization at the Library Scale (MUSCLE) combines single-molecule fluorescence microscopy with next-generation sequencing.
  • Ligation-based surface immobilization ensures high clustering efficiency (>40%) for increased throughput and simplified registration.
  • A 3D-printed flow cell adapter facilitates liquid exchange during single-molecule fluorescence microscopy.

Main Results:

  • MUSCLE enables highly multiplexed observations of complex dynamics on millions of individual molecules.
  • The method was successfully applied to study sequence-dependent dynamics of Cas9-induced target DNA unwinding and rewinding.
  • Demonstrated applicability to protein-nucleic acid interactions beyond nucleic-acid-only systems.

Conclusions:

  • MUSCLE provides a comprehensive dataset characterizing the dynamic behavior of molecular libraries.
  • This technique significantly enhances the throughput and scope of single-molecule analysis.
  • MUSCLE is a valuable tool for investigating complex biological phenomena involving diverse molecular entities.