Technique Merges Single-Molecule Imaging with Genomic Sequencing

Uppsala University researchers have developed a new technique that combines single-molecule fluorescence microscopy with next-generation sequencing, potentially transforming the study of complex biological processes. The technique, named MUSCLE (MUltiplexed Single-molecule Characterization at the Library scalE), allows scientists to observe and analyze the dynamics of individual molecules across vast libraries simultaneously.

Led by Professor Sebastian Deindl, the team overcame the limitations of traditional single-molecule fluorescence microscopy, which was restricted by low throughput. MUSCLE utilizes an Illumina MiSeq flow cell to attach fluorescently labeled molecules, which are then observed under a microscope using a 3D-printed adapter. Subsequently, the flow cell undergoes standard Illumina sequencing, and the resulting data is spatially matched with the previously observed molecules.

This innovative approach, described in a recent Science paper, enables researchers to profile the dynamics of numerous samples concurrently, providing a more comprehensive understanding of complex biological processes. "Our method allows for the direct observation of dynamic molecular behaviours across extensive libraries, significantly enhancing our ability to uncover general trends, outlier behaviours, and unique dynamic signatures that would otherwise remain hidden," explains Professor Deindl.

The team applied MUSCLE to study DNA hairpin dynamics and Cas9-induced DNA unwinding/rewinding, revealing unexpected behaviors in certain target sequences. This demonstrates the method's potential to uncover new biological insights and its broad applications across molecular biology, genetics, and drug discovery.

The accessibility of MUSCLE to the wider scientific community is a key advantage, as it relies on widely available equipment and can be easily adapted to study various proteins interacting with nucleic acids, as well as DNA-barcoded proteins, compounds, or ligands.

This new technique could help deepen our understanding of how nucleic acid-interacting proteins function in both health and disease.