Single-Molecule Imaging of DNA-Protein Interactions

Single-molecule imaging has revolutionised our understanding of how proteins engage with DNA by allowing direct visualisation of individual molecular events. Instead of averaging over large ensembles, researchers can now monitor the binding, sliding, and dissociation of single protein molecules on extended DNA substrates in real time. Techniques such as total internal reflection fluorescence microscopy (TIRFM), optical tweezers and nano-fabricated flow-stretch assays have been paired with precise surface chemistry to immobilise and extend DNA chains. These approaches have provided unprecedented insights into mechanisms of replication, transcription, repair and chromatin remodelling. By combining site-specific sequence modifications with advanced labelling strategies, it is possible to track the behaviour of molecular machines at defined locations, reveal transient intermediates and quantify kinetic parameters. The capacity to manipulate individual DNA–protein interactions under controlled forces and buffer conditions has enabled the dissection of fundamental processes that underpin genome maintenance and regulation, with implications for drug discovery and nanoscale engineering.

Research from Nature Portfolio

Recent studies have introduced a versatile molecular toolkit for rapid, high-yield insertion of recombinant sequences and chemical labels into long DNA substrates. This system leverages PCR-generated cassettes and a refined nicking-enzyme strategy to introduce extra-helical structures and functional groups at user-defined sites. As a demonstration, investigators examined the loading and translocation of replication clamp proteins on tailored DNA flaps and repeat motifs, uncovering structural features that influence clamp trapping and triplet-repeat expansion. In parallel, novel microfabricated surfaces combining hydrophobic and hydrophilic regions have been devised to perform high-density molecular combing followed by sequence-specific enzymatic fluorescent labelling. By controlling DNA extension and enzymatic access on the same substrate, this platform achieves large-scale, high-resolution mapping of linearised DNA, facilitating detailed studies of structural variants and enabling downstream single-molecule fluorescence assays under optimised conditions.

Single-Molecule Imaging of DNA-Protein Interactions publication trend

The graph below shows the total number of articles in single-molecule imaging of dna-protein interactions across all publications each year (not limited to Nature Index journals).

Technical terms

Single-molecule fluorescence imaging: A technique that uses fluorescent labels and sensitive microscopy to detect and track individual biomolecules in real time.

Molecular combing: A method for stretching and aligning DNA molecules on a surface by applying a receding meniscus, enabling high-resolution analysis.

DNA curtains: Organised arrays of extended DNA molecules on a lipid or patterned surface, allowing simultaneous high-throughput observation of protein interactions along many DNA strands.

Nicking enzyme: An endonuclease that introduces single-strand breaks at specific recognition sites, used for site-specific labelling or modification of DNA.

Locked nucleic acid oligomer: A modified nucleic acid probe with a constrained ribose ring, providing enhanced binding affinity and mechanical stability when hybridised to DNA.

References

1. Efficient modification of λ-DNA substrates for single-molecule studies. Scientific Reports (2017).
2. A micropatterned substrate for on-surface enzymatic labelling of linearized long DNA molecules. Scientific Reports (2019).
3. Oriented Soft DNA Curtains for Single-Molecule Imaging. Langmuir (2021).
4. Locked nucleic acid oligomers as handles for single molecule manipulation. Nucleic Acids Research (2014).
5. Real-time single-molecule visualization using DNA curtains reveals the molecular mechanisms underlying DNA repair pathways. DNA Repair (2023).