Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding

Rieu et al. present a magnetic tweezers based single-molecule manipulation method, called kinetic locking, for direct detection of biomolecular binding without use of fluorescent probes. By measuring dynamical interactions of E. coli RecQ helicase with its DNA substrate, authors show that this method holds promise for studying DNA-DNA and DNA-protein interactions while avoiding the need for labelling. Fluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein's natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5, 6, 7 bases) and use it to measure the dynamical interactions of Escherichia coli/E. coli RecQ helicase with its DNA substrate.

Automated summary

The researchers introduced a single-molecule manipulation method called kinetic locking based on magnetic tweezers, which enables direct detection of biomolecular binding without the use of fluorescent probes. By measuring the dynamic interactions of E. coli RecQ helicase with its DNA substrate, they demonstrated the potential of this method for studying DNA-DNA and DNA-protein interactions while avoiding the need for labeling. This fluorescence-free micro-manipulation technique allows functional characterization of DNA/RNA processing proteins, without interference from labels.