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Updated: Feb 12, 2026
16:05
Large-Scale Screens of Metagenomic Libraries
Published on: May 28, 2007
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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.
Nature Protocols
|June 4, 2025
Summary
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.
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.
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